Rapamycin formulations and methods of their use

ABSTRACT

Described herein are liquid rapamycin formulations. Described herein are methods of treating or preventing diseases or conditions, such as choroidal neovascularization, wet AMD and dry AMD, and preventing transition of dry AMD to wet AMD, using the liquid rapamycin formulations described herein.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 11/352,092, filed Feb. 9, 2006, which is related to and claimspriority from U.S. Provisional Patent Application No. 60/664,040, filedMar. 21, 2005, U.S. Provisional Patent Application No. 60/664,306, filedMar. 21, 2005, and U.S. Provisional Patent Application No. 60/651,790,filed Feb. 9, 2005, each of which is incorporated herein by reference inits entirety for all purposes.

FIELD

Described herein are liquid rapamycin formulations for treatment orprevention of age-related macular degeneration (AMD), by delivery of theliquid rapamycin formulation to the eye of a subject, including but notlimited to a human subject.

BACKGROUND

The retina of the eye contains the cones and rods that detect light. Inthe center of the retina is the macula lutea, which is about ⅓ to ½ cmin diameter. The macula provides detailed vision, particularly in thecenter (the fovea), because the cones are higher in density. Bloodvessels, ganglion cells, inner nuclear layer and cells, and theplexiform layers are all displaced to one side (rather than restingabove the cones), thereby allowing light a more direct path to thecones.

Under the retina are the choroid, comprising a collection of bloodvessels embedded within a fibrous tissue, and the deeply pigmentedepithelium, which overlays the choroid layer. The choroidal bloodvessels provide nutrition to the retina (particularly its visual cells).

There are a variety of retinal disorders for which there is currently notreatment or for which the current treatment is not optimal. Maculardegeneration and choroidal neovascularization are nonlimiting examplesof retinal disorders that are difficult to treat with conventionaltherapies.

Age-related macular degeneration (AMD) is the major cause of severevisual loss in the United States for individuals over the age of 60. AMDoccurs in either an atrophic or less commonly an exudative form. Theatrophic form of AMD is also called “dry AMD,” and the exudative form ofAMD is also called “wet AMD.”

In exudative AMD, blood vessels grow from the choriocapillaris throughdefects in Bruch's membrane, and in some cases the underlying retinalpigment epithelium. Organization of serous or hemorrhagic exudatesescaping from these vessels results in fibrovascular scarring of themacular region with attendant degeneration of the neuroretina,detachment and tears of the retinal pigment epithelium, vitreoushemorrhage and permanent loss of central vision. This process isresponsible for more than 80% of cases of significant visual loss insubjects with AMD. Current or forthcoming treatments include laserphotocoagulation, photodynamic therapy, treatment with pegylatedaptamers, treatment with VEGF antibody fragments, and treatment withcertain small molecule agents.

Several studies have recently described the use of laserphotocoagulation in the treatment of initial or recurrent neovascularlesions associated with AMD (Macular Photocoagulation Study Groups(1991) in Arch. Ophthal. 109:1220; Arch. Ophthal. 109:1232; and Arch.Ophthal. 109:1242). Unfortunately, AMD subjects with subfoveal lesionssubjected to laser treatment experienced a rather precipitous reductionin visual acuity (mean 3 lines) at 3 months follow-up. Moreover, at twoyears post-treatment treated eyes had only marginally better visualacuity than their untreated counterparts (means of 20/320 and 20/400,respectively). Another drawback of the procedure is that vision aftersurgery is immediately worse.

Photodynamic therapy (PDT) is a form of phototherapy, a termencompassing all treatments that use light to produce a beneficialreaction in a subject. Optimally, PDT destroys unwanted tissue whilesparing normal tissue. Typically, a compound called a photosensitizer isadministered to the subject. Usually, the photosensitizer alone haslittle or no effect on the subject. When light, often from a laser, isdirected onto a tissue containing the photosensitizer, thephotosensitizer is activated and begins destroying targeted tissue.Because the light provided to the subject is confined to a particularlytargeted area, PDT can be used to selectively target abnormal tissue,thus sparing surrounding healthy tissue. PDT is currently used to treatretinal diseases such as AMD. PDT is currently the mainstay of treatmentfor subfoveal choroidal neovascularization in subjects with AMD(Photodynamic Therapy for Subfoveal Choroidal Neovascularization in AgeRelated Macular Degeneration with Verteporfin by TAP Study Group (1999)in Arch. Ophthalmol. 117:1329-1345).

Choroidal neovascularization (CNV) has proven to be recalcitrant totreatment in most cases. Conventional laser treatment can ablate CNV andhelp to preserve vision in selected cases not involving the center ofthe retina, but this is limited to only about 10% of the cases.Unfortunately, even with successful conventional laser photocoagulation,the neovascularization recurs in about 50-70% of eyes (50% over 3 yearsand >60% at 5 years). (Macular Photocoagulation Study Group (1986) inArch. Ophthalmol. 204:694-701). In addition, many subjects who developCNV are not good candidates for laser therapy because the CNV is toolarge for laser treatment, or the location cannot be determined so thatthe physician cannot accurately aim the laser. Photodynamic therapy,although utilized in up to 50% of new cases of subfoveal CNV has onlymarginal benefits over natural history, and generally delays progressionof visual loss rather than improving vision which is already decreasedsecondary to the subfoveal lesion. PDT is neither preventive nordefinitive. Several PDT treatments are usually required per subject andadditionally, certain subtypes of CNV fare less well than others.

Thus, there remains a need for methods, compositions, and formulationsthat may be used to optimally prevent or significantly inhibit choroidalneovascularization and to prevent and treat AMD in its wet and dryforms.

In addition to AMD, choroidal neovascularization is associated with suchretinal disorders as presumed ocular histoplasmosis syndrome, myopicdegeneration, angioid streaks, idiopathic central serouschorioretinopathy, inflammatory conditions of the retina and or choroid,and ocular trauma. Angiogenic damage associated with neovascularizationoccurs in a wide range of disorders including diabetic retinopathy,venous occlusions, sickle cell retinopathy, retinopathy of prematurity,retinal detachment, ocular ischemia and trauma.

There have been many attempts to treat CNV and its related diseases andconditions, as well as other conditions such as macular edema andchronic inflammation, with pharmaceuticals. For example, use ofrapamycin to inhibit CNV and wet AMD has been described in U.S.application Ser. No. 10/665,203, which is incorporated herein byreference in its entirety. The use of rapamycin to treat inflammatorydiseases of the eye has been described in U.S. Pat. No. 5,387,589, thecontent of which is incorporated herein by reference in its entirety.U.S. Patent Application No. 60/503,840 and Ser. No. 10/945,682 arefurther incorporated herein by reference in their respective entireties.Another reference whose content is incorporated herein by reference inits entirety is U.S. Pat. No. 6,376,517.

Particularly for chronic diseases, including those described herein,there is a great need for long acting methods for delivering therapeuticagents to the eye, such as to the posterior segment to treat CNV in suchdiseases as AMD. Formulations with extended delivery of therapeuticagent are more comfortable and convenient for a subject, due to adiminished frequency of ocular injections of the therapeutic agent.

Direct delivery of therapeutic agents to the eye rather than systemicadministration may be advantageous because the therapeutic agentconcentration at the site of action is increased relative to thetherapeutic agent concentration in a subject's circulatory system.Additionally, therapeutic agents may have undesirable side effects whendelivered systemically to treat posterior segment disease. Thus,localized drug delivery may promote efficacy while decreasing sideeffects and systemic toxicity.

SUMMARY

The methods and liquid rapamycin formulations described herein allowdelivery of rapamycin to the eye of a subject. Unless the contextindicates otherwise, it is envisioned that the subjects on whom all ofthe methods of treatment may be performed include human subjects.

Described herein are methods and liquid rapamycin formulations fordelivering rapamycin for extended periods of time which can be used forthe treatment, prevention, inhibition, delaying onset of, or causingregression of diseases and conditions including CNV, wet AMD, and dryAMD.

As described in further detail in the Detailed Description section, themethods and liquid rapamycin formulations may also be used for deliveryto a subject or to the eye of a subject of therapeutically effectiveamounts of rapamycin for the treatment, prevention, inhibition, delayingof the onset of, or causing the regression of wet or dry AMD. In somevariations, the methods, compositions, and liquid formulations are usedto treat wet AMD. In some variations, the methods, compositions, andliquid formulations are used to prevent wet AMD. In some variations, themethods, compositions, and liquid formulations are used to treat dryAMD. In some variations, the methods, compositions, and liquidformulations are used to prevent dry AMD. In some variations, themethods, compositions, and liquid formulations are used to preventtransition from dry AMD to wet AMD. The methods, compositions and liquidformulations may also be used for delivery to a subject or to the eye ofa subject of therapeutically effective amounts of rapamycin for thetreatment, prevention, inhibition, delaying of the onset of, or causingthe regression of CNV. In some variations, the methods, compositions andliquid formulations are used to treat CNV. The methods, compositions andliquid formulations may also be used for delivery to a subject or to theeye of a subject of therapeutically effective amounts of rapamycin forthe treatment, prevention, inhibition, delaying of the onset of, orcausing the regression of angiogenesis in the eye. In some variations,the methods, compositions and liquid formulations are used to treatangiogenesis. Other diseases and conditions that may be treated,prevented, inhibited, have onset delayed, or caused to regress usingrapamycin are described in the Diseases and Conditions section of theDetailed Description.

In some variations, the liquid formulations described herein form anon-dispersed mass when placed into a rabbit eye, including but notlimited to the vitreous of a rabbit eye.

The liquid rapamycin formulations may generally be administered in anyvolume that has the desired effect. In some variations a volume of aliquid rapamycin formulation is administered to the vitreous and theliquid formulation is less than one half the volume of the vitreous. Insome variations, formation of a non-dispersed mass after placement ofthe liquid rapamycin formulation in a rabbit eye or a subject dependsupon the volume of the liquid rapamycin formulation injected or placedin the rabbit eye or subject. The liquid rapamycin formulationsdescribed herein are generally administered intraocularly, periocularly,intravitreally, or between the sclera and conjunctiva.

The liquid rapamycin formulations described herein may deliver rapamycinfor an extended period of time. One nonlimiting example of such anextended release delivery system is a liquid rapamycin formulation thatdelivers rapamycin to the eye of human a subject in an amount sufficientto treat, prevent, inhibit, delay onset of, or cause regression of wetor dry AMD, or CNV, in a subject for an extended period of time. In somevariations, the liquid rapamycin formulation is used to treat wet or dryAMD or CNV in a human subject. In some variations, the liquid rapamycinformulation is used to prevent transition of dry AMD to wet AMD in ahuman subject. In some variations, the liquid rapamycin formulationdelivers an amount of rapamycin effect to treat or prevent wet or dryAMD or CNV for at least about one, about two, about three, about six,about nine, or about twelve months. Other extended periods of releaseare described in the Detailed Description.

Described herein is a liquid formulation comprising about 2% (w/w) ofrapamycin, about 94% (w/w) PEG 400, and about 4% (w/w) of ethanol.Described herein is a method for treating wet age-related maculardegeneration in a human subject, the method comprising administering tothe human subject by intraocular or periocular delivery a volume of aliquid formulation comprising about 2% (w/w) of rapamycin, about 94%(w/w) PEG 400, and about 4% (w/w) of ethanol containing an amount ofrapamycin effective to treat wet age-related macular degeneration in thehuman subject. Described herein is a method for preventing wetage-related macular degeneration in a human subject, the methodcomprising administering to the human subject by intraocular orperiocular delivery a volume of a liquid formulation comprising about 2%(w/w) of rapamycin, about 94% (w/w) PEG 400, and about 4% (w/w) ofethanol containing an amount of rapamycin effective to prevent wetage-related macular degeneration in the human subject. In somevariations the human subject is identified as being at heightened riskof developing wet age-related macular degeneration in the eye to whichthe liquid formulation is administered. In some variations the humansubject has dry age-related macular degeneration in at least one eye. Insome variations the human subject has wet age-related maculardegeneration in one eye and the liquid formulation is administered tothe eye without wet age-related macular degeneration.

Described herein are methods for treating dry age-related maculardegeneration in a human subject comprising administering to the humansubject by intraocular or periocular delivery a volume of a liquidformulation comprising about 2% (w/w) of rapamycin, about 94% (w/w) PEG400, and about 4% (w/w) of ethanol.

Described herein are methods for preventing wet age-related maculardegeneration in a human subject having dry age-related maculardegeneration, the method comprising administering to a human subjecthaving dry age-related macular degeneration a volume of a liquidformulation comprising about 2% (w/w) of rapamycin, about 94% (w/w) PEG400, and about 4% (w/w) of ethanol, wherein the volume is administeredby intraocular or periocular delivery.

In some variations, the volume of a liquid formulation is administeredto the human subject by placement in the vitreous and the volume ofliquid formulation contains less than about 3 mg of rapamycin. In somevariations, the volume of liquid formulation contains less than about2.5 mg of rapamycin. In some variations, the volume of liquidformulation contains less than about 2 mg of rapamycin. In somevariations, the volume of liquid formulation contains between about 20μg and about 2.5 mg of rapamycin. In some variations, the volume ofliquid formulation is administered to the human subject by placementbetween the sclera and conjunctiva and the volume of liquid formulationcontains less than about 5 mg of rapamycin. In some variations, thevolume of liquid formulation contains less than about 3.5 mg ofrapamycin. In some variations, the volume of liquid formulation containsless than about 3 mg of rapamycin. In some variations, the volume ofliquid formulation contains between about 20 μg and about 5 mg ofrapamycin.

In some variations, the volume of liquid formulation is administered tothe human subject by placement in the vitreous of the human subject andthe volume of liquid formulation contains less than about 100 μL of PEG400. In some variations, the volume of liquid formulation contains lessthan about 50 μL of PEG 400. In some variations, wherein the volume ofliquid formulation contains less than about 30 μL of PEG 400.

In some variations, the volume of liquid formulation is administered tothe human subject by placement between the sclera and conjunctiva andthe volume of liquid formulation contains less than about 160 μL of PEG400. In some variations, the volume of liquid formulation contains lessthan about 120 μL of PEG 400. In some variations, wherein the volume ofliquid formulation contains less than about 90 μL of PEG 400.

In some variations, a volume of a liquid formulation described herein ofless than about 50 μL of liquid formulation is administered to the humansubject by placement in the vitreous of the human subject. In somevariations, a volume of less than about 20 μL of liquid formulation isadministered to the human subject. In some variations, a volume of lessthan about 10 μL of liquid formulation is administered to the humansubject. In some variations, a volume of less than about 5 μL of liquidformulation is administered to the human subject. In some variations, avolume of less than about 1 μL of liquid formulation is administered tothe human subject.

In some variations, a volume of less than about 200 μL of liquidformulation is administered to the human subject by placement betweenthe sclera and conjunctiva of the human subject. In some variations, avolume of less than about 100 μL of liquid formulation is administeredto the human subject. In some variations, a volume of less than about 50μL of liquid formulation is administered to the human subject. In somevariations, a volume of less than about 20 μL of liquid formulation isadministered to the human subject. In some variations, a volume of lessthan about 10 μL of liquid formulation is administered to the humansubject. In some variations, a volume of less than about 5 μL of liquidformulation is administered to the human subject.

In some variations, a volume of a liquid formulation is administered tothe human subject by placement between the sclera and conjunctiva andthe human subject to which the volume is administered has visual acuityof at least about 20/40. In some variations, the human subject to whichthe volume is administered has visual acuity of at least about 20/40 inthe eye to which the volume is administered.

Described herein are liquid formulations comprising rapamycin, anon-aqueous liquid component, and optionally a water component, whereinthe rapamycin is at least about 0.1% (w/w) of the liquid formulation andthe non-aqueous liquid component is at least about 90% (w/w) of theliquid formulation; and wherein the liquid formulation when injectedinto the vitreous of a rabbit eye delivers an amount of rapamycinsufficient to achieve an average concentration of rapamycin in theretina choroid of the rabbit eye of at least about 0.01 ng/mg for aperiod of time of at least about 30 or at least about 120 days followingadministration of the liquid formulation. In some variations, the liquidformulation when injected into the vitreous of a rabbit eye delivers anamount of rapamycin sufficient to achieve an average concentration ofrapamycin in the retina choroid of the rabbit eye of at least about 0.1ng/mg for a period of time of at least about 30 or at least about 90days following administration of the liquid formulation.

Described herein are liquid formulations comprising rapamycin, anon-aqueous liquid component, and optionally a water component, whereinthe rapamycin is at least about 0.1% (w/w) of the liquid formulation andthe non-aqueous liquid component is at least about 90% (w/w) of theliquid formulation; and wherein the liquid formulation when injectedinto the vitreous of a rabbit eye delivers an amount of rapamycinsufficient to achieve an average concentration of rapamycin in thevitreous of the rabbit eye of at least about 1000 ng/ml for a period oftime of at least about 30 or at least about 120 days followingadministration of the liquid formulation.

Described herein are liquid formulations comprising rapamycin, anon-aqueous liquid component, and optionally a water component, whereinthe rapamycin is at least about 0.1% (w/w) of the liquid formulation andthe non-aqueous liquid component is at least about 90% (w/w) of theliquid formulation; and wherein the liquid formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers an amount ofrapamycin sufficient to achieve an average concentration of rapamycin inthe vitreous of the rabbit eye of at least about 0.01 ng/ml for a periodof time of at least about 30 or at least about 120 days followingadministration of the liquid formulation. In some variations, the liquidformulation when injected between the sclera and conjunctiva of a rabbiteye delivers an amount of rapamycin sufficient to achieve an averageconcentration of rapamycin in the vitreous of the rabbit eye of at leastabout 0.1 ng/ml for a period of time of at least about 30 or at leastabout 120 days following administration of the liquid formulation.

Described herein are liquid formulations comprising rapamycin, anon-aqueous liquid component, and optionally a water component, whereinthe rapamycin is at least about 0.1% (w/w) of the liquid formulation andthe non-aqueous liquid component is at least about 90% (w/w) of theliquid formulation; and wherein the liquid formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers an amount ofrapamycin sufficient to achieve an average concentration of rapamycin inthe retina choroid of the rabbit eye of at least about 0.001 ng/mg for aperiod of time of at least about 30 or at least about 120 days followingadministration of the liquid formulation. In some variations, the liquidformulation when injected between the sclera and conjunctiva of a rabbiteye delivers an amount of rapamycin sufficient to achieve an averageconcentration of rapamycin in the retina choroid of the rabbit eye of atleast about 0.005 ng/mg for a period of time of at least about 30 or atleast about 120 days following administration of the liquid formulation.In some variations, the liquid formulation when injected between thesclera and conjunctiva of a rabbit eye delivers an amount of rapamycinsufficient to achieve an average concentration of rapamycin in theretina choroid of the rabbit eye of at least about 0.01 ng/mg for aperiod of time of at least about 30 days following administration of theliquid formulation.

Described herein are liquid formulations wherein the rapamycin is lessthan about 6% (w/w) of the liquid formulation, the water component isless than about 5% (w/w) of the liquid formulation, and the non-aqueousliquid component is selected from the group consisting of any one ormore of glycerin, dimethylsulfoxide, N-methylpyrrolidone, dimethylacetamide (DMA), dimethyl formamide, glycerol formal, ethoxy diglycol,triethylene glycol dimethyl ether, triacetin, diacetin, corn oil, acetyltriethyl citrate (ATC), ethyl lactate, polyglycolated capryl glyceride,γ butyrolactone, dimethyl isosorbide, or benzyl alcohol.

Described herein are unit dosage forms comprising a volume of a liquidformulation as described herein, wherein the volume of liquidformulation contains less than about 4 mg, less than about 3.5 mg, lessthan about 3 mg, less than about 2.5 mg, less than about 2 mg, betweenabout 20 μg and about 2.5 mg, or between about 20 μg and about 5 mg ofrapamycin.

Described herein are unit dosage forms comprising a volume of a liquidformulation as described herein, wherein the non-aqueous liquidcomponent is polyethylene glycol and the volume of liquid formulationcontains less than about 160 μL, less than about 120 μL, less than about90 μL, less than about 50 μL of polyethylene glycol, or less than about30 μL of polyethylene glycol.

Described herein are unit dosage forms comprising a volume of less thanabout 200 μL, less than about 100 μL, less than about 50 μL, less thanabout 20 μL, less than about 10 μL, less than about 5 μL, or less thanabout 1 μL of a liquid formulation described herein.

Described herein are methods for treating wet age-related maculardegeneration in a human subject, the method comprising administering tothe human subject by intraocular or periocular delivery a volume of aliquid formulation described herein containing an amount of rapamycineffective to treat wet age-related macular degeneration in the humansubject.

Described herein are methods for preventing wet age-related maculardegeneration in a human subject, the method comprising administering tothe human subject by intraocular or periocular delivery a volume of theliquid formulation described herein containing an amount of rapamycineffective to prevent wet age-related macular degeneration in the humansubject. In some variations the human subject is identified as being atheightened risk of developing wet age-related macular degeneration inthe eye to which the liquid formulation is administered. In somevariations the human subject identified as being at heightened risk ofdeveloping wet age-related macular degeneration has dry age-relatedmacular degeneration in at least one eye. In some variations the humansubject identified as being at heightened risk of developing wetage-related macular degeneration has wet age-related maculardegeneration in one eye and the liquid formulation is administered tothe eye without wet age-related macular degeneration.

Described herein are methods for treating dry age-related maculardegeneration in a human subject, the method comprising administering tothe human subject by intraocular or periocular delivery a volume of aliquid formulation described herein containing an amount of rapamycineffective to treat dry age-related macular degeneration in the humansubject.

Described herein are methods for preventing wet age-related maculardegeneration in a human subject having dry age-related maculardegeneration, the method comprising administering to a human subjecthaving dry age-related macular degeneration a volume of a liquidformulation described herein containing an amount of rapamycin effectiveto prevent wet age-related macular degeneration in the human subject,wherein the volume of the liquid formulation is administered byintraocular or periocular delivery.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C schematically depict formation of a non-dispersed mass,after injection of a liquid formulation into the vitreous of an eye, asit is believed to occur in some variations.

FIG. 2 depicts the level of rapamycin in the vitreous of rabbit eyes(ng/ml) at 5, 30, 60, 90, and 120 days after subconjunctival injectionof 20 μl, 40 μl, and 60 μl doses of a 2% solution of rapamycin inethanol and PEG 400.

FIG. 3 depicts the level of rapamycin in the retina choroid tissues ofrabbit eyes (ng/mg) at 5, 30, 60, 90, and 120 days after subconjunctivalinjection of 20 μl, 40 μl, and 60 doses of a 2% solution of rapamycin inethanol and PEG 400.

FIG. 4 depicts the level of rapamycin in the vitreous of rabbit eyes(ng/ml) at 5, 30, 60, 90, and 120 days after intravitreal injection of20 μl and 40 μl doses of a 2% solution of rapamycin in ethanol and PEG400.

FIG. 5 depicts the level of rapamycin in the retina choroid tissues ofrabbit eyes (ng/mg) at 5, 30, 60, 90, and 120 days after intravitrealinjection of 20 μl and 40 μl doses of a 2% solution of rapamycin inethanol and PEG 400.

DETAILED DESCRIPTION

Described in this section are liquid rapamycin formulations and methodsrelating to delivery of rapamycin to a subject or to the eye of asubject. These liquid rapamycin formulations and methods may be used forthe treatment, prevention, inhibition, delaying onset of, or causingregression of diseases and conditions of the eye including but notlimited to choroidal neovascularization; macular degeneration andage-related macular degeneration, including wet AMD and dry AMD. In somevariations, the liquid rapamycin formulations and methods are used fortreatment of choroidal neovascularization; macular degeneration andage-related macular degeneration, including wet AMD and dry AMD. In somevariations, the liquid rapamycin formulations and methods are used forprevention of choroidal neovascularization; macular degeneration andage-related macular degeneration, including wet AMD and dry AMD.

In this detailed description section are described (1) liquid rapamycinformulations, (2) extended delivery of rapamycin, (3) routes ofadministration for delivery of liquid rapamycin formulations, and (4)treatment and prevention of CNV and wet and dry AMD by delivery ofrapamycin to a subject or to the eye of a subject for an extended periodof time using the described liquid rapamycin formulations.

The term “about,” as used herein, generally refers to the level ofaccuracy that is obtained when the methods described herein, such as themethods in the examples, are used. However, by “about” a certain amountof a component of a formulation is meant 90-110% of the amount stated.

Liquid Rapamycin Formulations

The terms rapamycin and rapa are used interchangeably herein with theterm sirolimus. In some variations the liquid rapamycin formulationsform a non-dispersed mass relative to a surrounding medium when placedin the vitreous of a rabbit eye.

The liquid formulations described herein contain rapamycin and maygenerally be any liquid formulation, including but not limited tosolutions, suspensions, and emulsions.

The liquid rapamycin formulations may generally be administered in anyvolume that has the desired effect; in some variations a liquidrapamycin formulation is administered to the vitreous and the liquidrapamycin formulation is less than one half the volume of the vitreousof the eye to which it is being administered. In some variations theliquid rapamycin formulation is administered between the sclera andconjunctiva in a volume less than about 50 μl.

When a certain volume is administered, it is understood that there issome imprecision in the accuracy of various devices that may be used toadminister the liquid formulation. Where a certain volume is specified,it is understood that this is the target volume. However, certaindevices such as insulin syringes are inaccurate to greater than 10%, andsometimes inaccurate to greater than 20% or more. Hamilton HPLC typesyringes are generally considered precise to within 10%, and arerecommended for volumes at or below 10 μl.

In some variations, a volume of a liquid rapamycin formulation describedherein is administered to the vitreous of a human subject's eye that isless than about 500 μl, less than about 400 μl, less than about 300 μl,less than about 200 μl, less than about 100 μl, less than about 90 μl,less than about 80 μl, less than about 70 μl, less than about 60 μl,less than about 50 μl, less than about 40 μl, less than about 30 μl,less than about 20 μl, less than about 10 μl, less than about 5 μl, lessthan about 3 μl, or less than about 1 μl. In some variations, a volumeof a liquid rapamycin formulation described herein is administered tothe vitreous of a human subject's eye that is less than about 20 μl. Insome variations, a volume of a liquid rapamycin formulation describedherein is administered to the vitreous that is less than about 10 μl. Insome variations, a volume of a liquid rapamycin formulation describedherein is administered to the vitreous of a human subject's eye that isbetween about 0.1 μl and about 200 μl, between about 50 μl and about 200μl, between about 50 μl and about 150 μl, between about 0.1 μl and about100 μl, between about 0.1 μl and about 50 μl, between about 1 μl andabout 40 μl, between about 1 μl and about 30 μl, between about 1 μl andabout 20 μl, between about 1 μl and about 10 μl, or between about 1 μland about 5 μl. In some variations, a volume of a liquid rapamycinformulation described herein is administered to the vitreous of a humansubject's eye that is between about 1 μl and about 10 μl. In somevariations, a volume of a liquid rapamycin formulation described hereinis administered to the vitreous of a rabbit eye or a subject's eye thatis between about 1 μl and about 5 μl. In some variations, a volume of aliquid rapamycin formulation described herein is administered to thevitreous of a rabbit eye or a subject's eye that is between about 1 μland about 5 μl. In some variations, a volume of a liquid rapamycinformulation described herein is administered to the vitreous of a humansubject's eye that is between about 0.1 μl and about 200 μl.

In some variations, a total volume of a liquid rapamycin formulationdescribed herein is subconjunctivally administered to a human subject'seye that is less than about 1000 μl, less than about 900 μl, less thanabout 800 μl, less than about 700 μl, less than about 600 μl, less thanabout 500 μl, less than about 400 μl, less than about 300 μl, less thanabout 200 μl, less than about 100 μl, less than about 90 μl, less thanabout 80 μl, less than about 70 μl, less than about 60 μl, less thanabout 50 μl, less than about 40 μl, less than about 30 μl, less thanabout 20 μl, less than about 10 μl, less than about 5 μl, less thanabout 3 μl, or less than about 1 μl. In some variations, a volume of aliquid rapamycin formulation described herein is subconjunctivallyadministered to a human subject's eye that is less than about 20 μl. Insome variations, a volume of a liquid rapamycin formulation describedherein is subconjunctivally administered to a human subject's eye thatis less than about 10 μl. In some variations, a volume of a liquidrapamycin formulation described herein is subconjunctivally administeredto a human subject's eye that is between about 0.1 μl and about 200 μl,between about 50 μl and about 200 μl, between about 200 μl and about 300μl, between about 300 μl and about 400 μl, between about 400 μl andabout 500 μl, between about 500 μl and about 600 μl, between about 600μl and about 700 μl, between about 700 μl and about 800 μl, betweenabout 800 μl and about 900 μl, between about 900 μl and about 1000 μl,between about 50 μl and about 150 μl, between about 0.1 μl and about 100μl, between about 0.1 μl and about 50 μl, between about 1 μl and about40 μl, between about 1 μl and about 30 μl, between about 1 μl and about20 μl, between about 1 μl and about 10 μl, or between about 1 μl andabout 5 μl. In some variations, a volume of a liquid rapamycinformulation described herein is subconjunctivally administered to ahuman subject's eye that is between about 1 μl and about 10 μl. In somevariations, a volume of a liquid rapamycin formulation described hereinis subconjunctivally administered to a human subject's eye that isbetween about 1 μl and about 5 μl. In some variations, a volume of aliquid rapamycin formulation described herein is administered tosubconjunctivally administered to a human subject's eye that is betweenabout 1 μl and about 5 μl. In some variations, a volume of a liquidrapamycin formulation described herein is administered tosubconjunctivally administered to a human subject's eye that is betweenabout 0.1 μl and about 200 μl.

In some variations the liquid rapamycin formulations described hereincontain no greater than about 250 μl of polyethylene glycol. In somevariations the liquid rapamycin formulation described herein contain nogreater than about 250 μl, no greater than about 200 μl, no greater thanabout 150 μl, no greater than about 125 μl, no greater than about 100μl, no greater than about 75 μl, no greater than about 50 μl, no greaterthan about 25 μl, no greater than about 20 μl, no greater than about 15μl, no greater than about 10 μl, no greater than about 7.5 μl, nogreater than about 5 μl, no greater than about 2.5 μl, no greater thanabout 1.0 μl, or no greater than about 0.5 μl of polyethylene glycol.Formulations containing polyethylene glycol may contain, for example,PEG 300 or PEG 400.

In some variations, the liquid rapamycin formulation described hereinhave a viscosity of between 40% and 120% centipoise. In some variationsthe liquid rapamycin formulations described herein have a viscosity ofbetween 60% and 80% centipoise.

In some variations the liquid rapamycin formulations described hereinare administered in multiple subconjunctival locations within a periodof time of one another, including but not limited to within an hour ofone another. Without being bound by theory, it is thought that suchmultiple administrations, such as multiple injections, allow for agreater total dose to be administered subconjunctivally than a singledose due to a potentially limited ability of the local ocular tissues toabsorb larger volumes.

Some liquid rapamycin formulations described herein comprise anon-aqueous liquid component. The non-aqueous liquid component maycomprise a single non-aqueous liquid component or a combination ofnon-aqueous liquid component. In some variations, the non-aqueous liquidcomponent is glycerin, dimethylsulfoxide, N-methylpyrrolidone, ethanol,isopropyl alcohol, polyethylene glycol of various molecular weights,including but not limited to PEG 300 and PEG 400, or propylene glycol,or a mixture of one or more thereof.

Liquid rapamycin formulations may optionally further comprisestabilizers, excipients, gelling agents, adjuvants, antioxidants, and/orother components as described herein.

In some variations all components in the liquid formulation, other thanthe therapeutic agent, are liquid at room temperature.

In some variations the rapamycin in the liquid formulation containsbetween about 0.01 to about 10% of the total weight of the composition;between about 0.05 to about 10%; between about 0.1 to about 5%; betweenabout 1 to about 5%; or between about 5 to about 15%; between about 8 toabout 10%; between about 0.01 to about 1%; between about 0.05 to about5%; between about 0.1 to about 0.2%; between about 0.2 to about 0.3%;between about 0.3 to about 0.4%; between about 0.4 to about 0.5%;between about 0.5 to about 0.6%; between about 0.6 to about 0.7%;between about 0.7 to about 1%; between about 1 to about 3%; or betweenabout 1.5 to about 2.5%. In some variations the liquid formulationsdescribed herein contain between about 0.1 to about 5% w/w of rapamycin.

In some variations the non-aqueous liquid component is, by way ofnonlimiting example, between about 0.01 to about 99.9% of the totalweight of the composition; between about 0.1 to about 99%; between about75 to about 99.99%; between about 85 to about 99.99%; or between about55 to about 95% w/w. In some variations the non-aqueous liquid componentis between about 85 to about 99.99% w/w.

In some variations there is optionally a water component. In somevariations the water component is less than about 30%, less than about25%, less than about 20%, less than about 15%, less than about 10%, lessthan about 7.5%, less than about 5%, less than about 4%, less than about3%, less than about 2%, less than about 1%, or less than about 0.5%. Insome variations the water component is less than about 5% w/w.

Some variations of liquid formulations includes rapamycin between about0.01 and about 5% by weight of the total, and a non-aqueous liquidcomponent between about 95% and about 99.99% by weight of the total. Insome variations the formulations further comprise stabilizing agents,excipients, adjuvants, or antioxidants, between about 0 and about 5% byweight of the total.

In some variations, a liquid formulation may contain about 2% w/wrapamycin and about 98% w/w of a non-aqueous liquid component. In somevariations, the non-aqueous liquid component comprises ethanol. In somevariations, the non-aqueous liquid component comprises a liquidpolyethylene glycol, including but not limited to PEG 400.

Non-aqueous liquid components that may be used include but are notlimited to any non-aqueous liquid component as above, including but notlimited to any one or more of DMSO, glycerin, ethanol, methanol,isopropyl alcohol; castor oil, propylene glycol, polyvinylpropylene,polysorbate 80, benzyl alcohol, dimethyl acetamide (DMA), dimethylformamide (DMF), glycerol formal, ethoxy diglycol (Transcutol,Gattefosse), tryethylene glycol dimethyl ether (Triglyme), dimethylisosorbide (DMI), γ-butyrolactone, N-Methyl-2-pyrrolidinone (NMP),polyethylene glycol of various molecular weights, including but notlimited to PEG 300 and PEG 400, and polyglycolated capryl glyceride(Labrasol, Gattefosse).

Further non-aqueous liquid components include but are not limited toC₆-C₂₄ fatty acids, oleic acid, Imwitor 742, Capmul, F68, F68 (Lutrol),PLURONICS including but not limited to PLURONICS F108, F127, and F68,Poloxamers, Jeffamines), Tetronics, F127, beta-cyclodextrin, CMC,polysorbitan 20, Cavitron, softigen 767, captisol, and sesame oil.

Other methods that may be used to dissolve rapamycin are described inSolubilization of Rapamycin, Int'l J. Pharma 213 (2001) 25-29, thecontent of which is incorporated herein in its entirety.

As a nonlimiting example, rapamycin can be dissolved in 5% DMSO ormethanol in a balanced salt solution. The rapamycin solution can beunsaturated, a saturated or a supersaturated solution of rapamycin. Therapamycin solution can be in contact with solid rapamycin. In onenonlimiting example, rapamycin can be dissolved in a concentration of upto about 400 mg/ml. Rapamycin can also, for example, be dissolved inpropylene glycol esterified with fatty acids such as oleic, stearic,palmic, capric, linoleic, etc.

Many other non-aqueous liquid components are possible. Those of ordinaryskill in the art, given the teachings herein will find it routine toidentify non-aqueous liquid components for use in the liquid rapamycinformulations described herein.

Non-aqueous liquid components for use in the liquid formulations can bedetermined by a variety of methods known in the art, including but notlimited to (1) theoretically estimating their solubility parametervalues and choosing the ones that match with the therapeutic agent,using standard equations in the field; and (2) experimentallydetermining the saturation solubility of therapeutic agent in thenon-aqueous liquid components, and choosing the one(s) that exhibit thedesired solubility.

In some variations, the liquid rapamycin formulations form anon-dispersed mass when placed into an aqueous medium. As used herein, a“non-dispersed mass” refers to the structure formed when the liquidformulation is placed into an environment, relative to the environmentin which it is placed. Generally, a non-dispersed mass of a liquidformulation is anything other than a homogeneous distribution of theliquid formulation in the surrounding medium. The non-dispersed massmay, for instance, be indicated by visually inspecting the administeredliquid formulation and characterizing its appearance relative to thesurrounding medium.

In some variations, the aqueous medium is water. In some variations, thewater is deionized, distilled, sterile, or tap water, including but notlimited to tap water available at the place of business of MacuSight inUnion City, Calif.

In some variations, the aqueous medium is an aqueous medium of asubject. In some variations the aqueous medium is an aqueous medium ofthe eye of a subject, including but not limited to the vitreous of aneye of a subject. In some variations the subject is a human subject. Insome variations the aqueous medium is the vitreous of a rabbit eye.

The liquid formulations described herein may generally be of anygeometry or shape after administration to a subject or the eye of asubject. The non-dispersed mass-forming liquid formulations may, forinstance, appear as a compact spherical mass when administered to thevitreous. In other instances, the liquid formulation may appear as anon-dispersed mass relative to the surrounding medium, wherein thenon-dispersed mass is less clearly defined and the geometry is moreamorphous than spherical.

The non-dispersed mass-forming liquid formulations described herein mayform a non-dispersed mass immediately upon placement in the medium orthe non-dispersed mass may form some period of time after placement ofthe liquid formulation. In some variations the non-dispersed mass formsover the course of about 1, about 2, about 3, about 4, about 5, about 6,or about 7 days. In some variations the non-dispersed mass forms overthe course of about 1 week, about 2 weeks, or about 3 weeks.

In some variations the liquid formulations described herein form anon-dispersed mass which has the form of a solid depot when theformulation is injected into any or all of water, the vitreous, orbetween the sclera and the conjunctiva of a rabbit eye. In somevariations the liquid formulations described herein form a non-dispersedmass which has the form of a semi-solid when the formulation is injectedinto any or all of water, the vitreous, or between the sclera and theconjunctiva of a rabbit eye.

In some variations described herein the liquid rapamycin formulationforms a non-dispersed mass relative to a surrounding medium where thesurrounding medium is aqueous. An “aqueous medium” or “aqueousenvironment” is one that contains at least about 50% water. Examples ofaqueous media include but are not limited to water, the vitreous,extracellular fluid, conjunctiva, sclera, between the sclera and theconjunctiva, aqueous humor, gastric fluid, and any tissue or body fluidcomprised of at least about 50% of water. Aqueous media include but arenot limited to gel structures, including but not limited to those of theconjunctiva and sclera. In some variations described herein the liquidrapamycin formulation forms a non-dispersed mass when placed in thevitreous of a rabbit eye.

Whether a liquid formulation exhibits a non-dispersed mass relative to asurrounding medium when present in a subject or the eye of a subject maybe determined by, for instance, preparing the liquid rapamycinformulation, administering it to the vitreous of a rabbit eye, andcomparing the liquid formulation to the surrounding medium.

The liquid rapamycin formulations described herein may or may not form anon-dispersed mass in the subject. One liquid formulation describedherein forms a non-dispersed mass when administered to a subject andforms a non-dispersed mass when administered to a rabbit eye.

It is believed that the low solubility of rapamycin in the vitreouscontributes to the formation of a non-dispersed mass by somerapamycin-containing liquid formulations described herein. The vitreousis a clear gel composed almost entirely of water (up to 99%). Asrapamycin in an injected formulation contacts the vitreous, therapamycin precipitates.

Factors believed to affect the formation of and geometry of anon-dispersed mass include the concentration of rapamycin in theformulation, the viscosity of the formulation, ethanol content of theformulation, and the volume of injection. It is believed thatmaintaining a relatively high local concentration of rapamycin duringprecipitation favors formation of a non-dispersed mass. As volume isincreased for a given dose, formation of a non-dispersed mass may becomeless favorable. Formation of a non-dispersed mass may become morefavorable as rapamycin concentration is increased and/or as viscosity isincreased. Ethanol content affects both the solubility of the rapamycinin the formulation and the viscosity of the formulation.

Without being bound by theory, in some variations it is hypothesizedthat injection of certain volumes of a liquid formulation containingrapamycin, ethanol and polyethylene glycol results in formation of anon-dispersed mass as depicted in FIGS. 1A-1C and described as follows.Upon injection, due to its viscosity a solution forms a sphericalglobule 100 within the vitreous 110. Ethanol then diffuses out of thisglobule, resulting in localized precipitation 120 of the rapamycinwithin the globule. Eventually, the polyethylene glycol also diffusesout of the globule to leave a solid, compact non-dispersed mass ofrapamycin 130.

In some variations, upon formation a non-dispersed mass comprisingrapamycin, for example, delivers the drug continuously at approximatelya constant rate for an extended period of time. It is believed thatdelivery of rapamycin from a non-dispersed mass in the vitreous dependson dissolution of the rapamycin in the vitreous, which depends in turnon clearance of the drug from the vitreous to other tissues. Thisrelease process is believed to maintain a steady-state concentration ofrapamycin in the vitreous.

In some variations, formation of a non-dispersed mass reduces thetoxicity of the injected liquid formulation compared to an equivalentdose that did not form a non-dispersed mass. In variations in which aliquid formulation injected into the vitreous does not form anon-dispersed mass, the drug appears to disperse in the vitreous body.This can interfere with vision.

In some variations, it is believed that the liquid formulations willform a visually observable non-dispersed mass when injected into the eyeof a subject, including but not limited to a human subject.

In some variations, liquid formulations are believed to formnon-dispersed masses when injected subconjunctivally. In some variationsit is believed that when subconjunctivally administered the liquidformulation forms a depot in the scleral tissue. That is, it is believedthat the therapeutic agent is absorbed into the sclera proximate to theinjection site and forms a local concentration of drug in the sclera.

The compositions and liquid formulations described herein may be used todeliver amounts of rapamycin effective for treating, preventing,inhibiting, delaying on set of, or causing the regression of thediseases and conditions described herein. In some variations thecompositions and liquid formulations described herein deliver one ormore therapeutic agents over an extended period of time.

An “effective amount,” which is also referred to herein as a“therapeutically effective amount,” of rapamycin for administration asdescribed herein is that amount of rapamycin that provides thetherapeutic effect sought when administered to the subject. Theachieving of different therapeutic effects may require differenteffective amounts of rapamycin. For example, the therapeuticallyeffective amount of rapamycin used for preventing a disease or conditionmay be different from the therapeutically effective amount used fortreating, inhibiting, delaying the onset of, or causing the regressionof the disease or condition. In addition, the therapeutically effectiveamount may depend on the age, weight, and other health conditions of thesubject as is well know to those versed in the disease or conditionbeing addressed. Thus, the therapeutically effective amount may not bethe same in every subject to which the rapamycin is administered.

An effective amount of rapamycin for treating, preventing, inhibiting,delaying the onset of, or causing the regression of a specific diseaseor condition is also referred to herein as the amount rapamycineffective to treat, prevent, inhibit, delay the onset of, or cause theregression of the disease or condition.

To determine whether a level of rapamycin is a therapeutically effectiveamount to treat, prevent, inhibit, delay on set of, or cause theregression of the diseases and conditions described in the Diseases andConditions section, liquid formulations may be administered in animalmodels for the diseases or conditions of interest, and the effects maybe observed. Dose ranging clinical trials may be performed to determineeffective amounts.

The formulations described herein may further comprise various othercomponents such as stabilizers, adjuvants, anti-oxidants (e.g.,tocopherol, BHA, BHT, TBHQ, tocopherol acetate, ascorbyl palmitate,ascorbic acid propyl gallate, and the like), preservatives, or diluents,for example. Other components that may be used in the formulationsdescribed herein include but are not limited to agents that will (1)improve the compatibility of excipients with the encapsulating materialssuch as gelatin, (2) improve the stability (e.g. prevent crystal growthof a therapeutic agent such as rapamycin) of rapamycin, and/or (3)improve formulation stability. Note that there is overlap betweencomponents that are stabilizers and those that are non-aqueous liquidcomponents, and the same component can carry out more than one role.

The rapamycin may be subjected to conventional pharmaceuticaloperations, such as sterilization, and compositions containing rapamycinmay also contain conventional adjuvants, such as preservatives,stabilizers, wetting agents, emulsifiers, buffers etc. The liquidrapamycin formulation may also be formulated with pharmaceuticallyacceptable excipients for clinical use to produce a pharmaceuticalcomposition. The liquid rapamycin formulation may be used to prepare amedicament to treat, prevent, inhibit, delay onset, or cause regressionof any of the conditions described herein. In some variations, theliquid rapamycin formulation may be used to prepare a medicament totreat any of the conditions described herein.

The liquid rapamycin formulations may conveniently be presented in unitdosage form and may be prepared by conventional pharmaceuticaltechniques. Such techniques include the step of bringing intoassociation the therapeutic agent and the pharmaceutical carrier(s) orexcipient(s). The liquid rapamycin formulations may be prepared byuniformly and intimately bringing into associate the active ingredientwith liquid carriers or finely divided solid carriers or both, and then,if necessary, shaping the product. The unit dosage form may be ready forplacement or injection into the eye of a subject, or may be diluted inan aqueous or non-aqueous medium prior to injection or placement in theeye of the subject.

In some variations, the formulations described herein are provided inone or more unit dose forms, wherein the unit dose form contains anamount of a liquid rapamycin formulations described herein that iseffective to treat or prevent the disease or condition for which it isbeing administered.

In some variations, the unit dose form is prepared in the concentrationat which it will be administered. In some variations, the unit dose formis diluted prior to administration to a subject. In some variations, aliquid formulation described herein is diluted in an aqueous mediumprior to administration to a subject, including but not limited to anisotonic aqueous medium. In some variations, a liquid formulationdescribed herein is diluted in a non-aqueous medium prior toadministration to a subject.

In some variations provided herein are kits comprising one or more unitdose forms as described herein. In some embodiments, the kit comprisesone or more of packaging and instructions for use to treat one or morediseases or conditions. In some embodiments, the kit comprises a diluentwhich is not in physical contact with the formulation or pharmaceuticalformulation. In some embodiments, the kit comprises any of one or moreunit dose forms described herein in one or more sealed vessels. In someembodiments, the kit comprises any of one or more sterile unit doseforms.

In some variations, the unit dose form is in a container, including butnot limited to a sterile sealed container. In some variations thecontainer is a vial, ampule, or low volume applicator, including but notlimited to a syringe. In some variations, a low-volume applicator ispre-filled with rapamycin for treatment of an ophthalmic disease orcondition, including but not limited to a limus compound for treatmentof age-related macular degeneration. Described herein is a pre-filledlow-volume applicator pre-filled with a formulation comprisingrapamycin. In some variations a low-volume applicator is pre-filled witha solution comprising rapamycin and a polyethylene glycol, andoptionally further comprises one or more additional components includingbut not limited to ethanol. In some variations a low-volume applicatoris pre-filled with a solution comprising about 2% rapamycin, about 94%PEG-400, about 4% ethanol.

Described herein are kits comprising one or more containers. In somevariations a kit comprises one or more low-volume applicators pre-filledwith one or more formulations in liquid form comprising rapamycin,including but not limited to formulations in liquid form comprisingrapamycin, formulations in liquid form comprising rapamycin and apolyethylene glycol, and optionally further comprises one or moreadditional components including but not limited to ethanol, andformulations in liquid form comprising about 2% rapamycin, about 94%PEG-400, about 4% ethanol. In some variations the kit comprises one ormore containers, including but not limited to pre-filled low-volumeapplicators, with instructions for its use. In a further variation a kitcomprises one or more low-volume applicators pre-filled with rapamycin,with instructions for its use in treating a disease or condition of theeye.

In some variations, the containers described herein are in a secondarypackaging which limits exposure of the liquid rapamycin formulation tolight or oxygen.

The following references, each of which is incorporated herein byreference in its entirety, show one or more formulations, including butnot limited to rapamycin formulations, and which describe use ofrapamycin at various doses and other therapeutic agents for treatingvarious diseases or conditions: U.S. 60/651,790, filed Feb. 9, 2005;U.S. 60/664,040, filed Feb. 9, 2005; U.S. 60/664,119, filed Mar. 21,2005; U.S. 60/664,306, filed Mar. 21, 2005; U.S. Ser. No. 11/351,844,filed Feb. 9, 2006; U.S. Ser. No. 11/351,761, filed Feb. 9, 2006; US2005/0187241, and US 2005/0064010.

Extended Delivery of Rapamycin

Described herein are compositions and liquid formulations showing invivo delivery or clearance profiles with one or more of the followingcharacteristics. The delivery or clearance profiles are for clearance ofrapamycin in vivo after injection of the composition or liquidformulations subconjunctivally or into the vitreous of a rabbit eye. Thevolume of the rabbit vitreous is approximately 30-40% of the volume ofthe human vitreous. Not being bound by theory, it is estimated that thesurface area of the retina choroid of a rabbit eye is approximately 25%of the surface area of the retina choroid of a human eye. The amount ofrapamycin is measured using techniques as described in Example 3, butwithout limitation to the formulation described in Example 3.

The average concentration of rapamycin in the tissue of a rabbit eye ata given time after administration of a formulation containing rapamycinmay be measured according to the following method. Where volumes below10 μl are to be injected, a Hamilton syringe is used.

The liquid formulations are stored at a temperature of 2-8° C. prior touse.

The experimental animals are specific pathogen free (SPF) New ZealandWhite rabbits. A mixed population of about 50% male, about 50% female isused. The rabbits are at least 12 weeks of age, usually at least 14weeks of age, at the time of dosing. The rabbits each weigh at least 2.2kg, usually at least 2.5 kg, at the time of dosing. Prior to the study,the animals are quarantined for at least one week and examined forgeneral health parameters. Any unhealthy animals are not used in thestudy. At least 6 eyes are measured and averaged for a given time point.

Housing and sanitation are performed according to standard proceduresused in the industry. The animals are provided approximately 150 gramsof Teklad Certified Hi-Fiber Rabbit Diet daily, and are provided tapwater ad libitum. No contaminants are known to exist in the water and noadditional analysis outside that provided by the local water district isperformed. Environmental Conditions are monitored.

Each animal undergoes a pre-treatment ophthalmic examination (slit lampand ophthalmoscopy), performed by a board certified veterinaryophthalmologist. Ocular findings are scored according to the McDonaldand Shadduck scoring system as described in Dermatoxicology, F. N.Marzulli and H. I. Maibach, 1977 “Eye Irritation,” T. O. McDonald and J.A. Shadduck (pages 579-582). Observations are recorded using astandardized data collection sheet. Acceptance criteria for placement onstudy are as follows: scores of ≦1 for conjunctival congestion andswelling; scores of 0 for all other observation variables.

Gentamicin ophthalmic drops are placed into both eyes of each animaltwice daily on the day prior to dosing, on the day of dosing (Day 1),and on the day after dosing (Day 2). Dosing is performed in two phases,the first including one set of animals and the second including theother animals. Animals are randomized separately into masked treatmentgroups prior to each phase of dosing according to modified Latinsquares. Animals are fasted at least 8 hours prior to injection. Thestart time of the fast and time of injection are recorded.

Animals are weighed and anesthetized with an intravenous injection of aketamine/xylazine cocktail (87 mg/mL ketamine, 13 mg/mL xylazine) at avolume of 0.1-0.2 mL/kg. Both eyes of each animal are prepared forinjection as follows: approximately five minutes prior to injection,eyes are moistened with an ophthalmic Betadine solution. After fiveminutes, the Betadine is washed out of the eyes with sterile saline.Proparacaine hydrochloride 0.5% (1-2 drops) is delivered to each eye.For eyes to be intravitreally injected, 1% Tropicamide (1 drop) isdelivered to each eye.

On Day 1, both eyes of each animal receive an injection of test orcontrol article. Animals in selected groups are dosed a second time onDay 90±1. Dosing is subconjunctival or intravitreal. Actual treatments,injection locations, and dose volumes are masked and revealed at the endof the study.

Subconjunctival injections are given using an insulin syringe and 30gauge×½-inch needle. The bulbar conjunctiva in the dorsotemporalquadrant is elevated using forceps. Test article is injected into thesubconjunctival space.

Intravitreal injections are given using an Insulin syringe and 30gauge×½-inch needle. For each injection, the needle is introducedthrough the ventral-nasal quadrant of the eye, approximately 2-3 mmposterior to the limbus, with the bevel of the needle directed downwardand posteriorly to avoid the lens. Test article is injected in a singlebolus in the vitreous near the retina.

Animals are observed for mortality/morbidity twice daily. An animaldetermined to be moribund is euthanized with an intravenous injection ofcommercial euthanasia solution. Both eyes are removed and stored frozenat −70° C. for possible future evaluation. If an animal is found deadprior to onset of rigor mortis, both eyes are removed and stored frozenat −70° C. for possible future evaluation. Animals found after the onsetof rigor mortis are not necropsied.

Animals are weighed at randomization, on Day 1 prior to dosing, andprior to euthanasia.

Ophthalmic observations (slit lamp and indirect ophthalmoscopy) areperformed on all animals on Days 5±1, 30±1, 60±1, 90±1, and at laterdates in some variations. Observations are performed by a boardcertified veterinary ophthalmologist. For animals to be dosed on Day90±1, ophthalmic observations are performed prior to dosing. Ocularfindings are scored according to the McDonald and Shadduck scoringsystem as described in Dermatoxicology, F. N. Marzulli and H. I.Maibach, 1977 “Eye Irritation”, T. O. McDonald and J. A. Shadduck (pages579-582) and observations are recorded using a standardized datacollection sheet.

Whole blood samples (1-3 mL per sample) are collected from each animalprior to necropsy in vacutainer tubes containing EDTA. Each tube isfilled at least ⅔ full and thoroughly mixed for at least 30 seconds.Tubes are stored frozen until shipped on dry ice.

Animals are euthanized with an intravenous injection of commercialeuthanasia solution. Euthanasia is performed according to standardprocedures used in the industry.

For treatment groups dosed intravitreally or subconjunctivally withplacebo, all eyes from each of these groups are placed into Davidsonssolution for approximately 24 hours. Following the 24-hour period, theeyes are transferred to 70% ethanol; these globes are submitted formasked histopathological evaluation by a board certified veterinarypathologist. The time that eyes are placed into Davidsons and the timeof removal are recorded.

For treatment groups dosed intravitreally or subconjunctivally with testarticle, some eyes from each of these groups are frozen at −70° C. andsubmitted for pharmacokinetic analysis. The remaining eyes from each ofthese groups are placed into Davidsons solution for approximately 24hours. Following the 24-hour period, the eyes are transferred to 70%ethanol; these globes are submitted for masked histopathologicalevaluation by a board certified veterinary pathologist. The time thateyes are placed into Davidsons and the time of removal are recorded.

Frozen samples submitted for pharmacokinetic analysis are dissected withdisposable instruments. One set of instruments is used per eye, and thendiscarded. The samples are thawed at room temperature for 1 to 2 minutesto ensure that the frost around the tissue has been removed. The sclerais dissected into 4 quadrants, and the vitreous is removed. If anon-dispersed mass (NDM) is clearly visible within the vitreous, thevitreous is separated into two sections. The section with the NDM isapproximately two-thirds of the vitreous. The section without the NDM isthe portion of the vitreous that is the most distant from the NDM. Theaqueous humor, lens, iris, and cornea are separated. The retina choroidtissue is removed using a forceps and collected for analysis. Theconjunctiva is separated from the sclera.

The various tissue types are collected into separate individualpre-weighed vials which are then capped and weighed. The vials of tissueare stored at −80° C. until analyzed.

The sirolimus content of the retina choroid, sclera, vitreous humor, andwhole anti-coagulated blood is determined by high-pressure liquidchromatography/tandem mass spectroscopy (HPLC/MS/MS) using32-O-desmethoxyrapamycin as an internal standard. Where an NDM wasobserved in the vitreous, the section of the vitreous containing the NDMand the section of the vitreous not containing the NDM are analyzedseparately.

The average concentration of rapamycin over a period of time means forrepresentative timepoints over the period of time the averageconcentration at each time point. For example, if the time period is 30days, the average concentration may be measured at 5 day intervals: forthe average concentration at day 5, the average of a number ofmeasurements of concentration at day 5 would be calculated; for theaverage concentration at day 10, the average of a number of measurementsof the concentration at day 10 would be calculated, etc.

In some variations, the liquid formulations described herein may have invivo delivery to the vitreous profiles with the following describedcharacteristics, where the delivery profiles are for delivery ofrapamycin in vivo after injection of the liquid formulation between thesclera and the conjunctiva of a rabbit eye. One nonlimiting variation ofin vivo delivery to the vitreous profiles is shown in FIG. 2.

“Approximately constant,” as used herein, means that the average leveldoes not vary by more than one order of magnitude over the extendedperiod of time, i.e., the difference between the maximum and minimum isless than a 10-fold difference for measurements of the averageconcentration at times in the relevant period of time.

In some variations, the liquid rapamycin formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers rapamycingiving an average concentration of rapamycin in the vitreous of therabbit eye of at least 0.001 ng/mL for at least 30, at least 60, atleast 90, or at least 120 days after administration of the liquidrapamycin formulation to the rabbit eyes. In some variations, the liquidrapamycin formulation when injected between the sclera and conjunctivaof a rabbit eye delivers rapamycin giving an average concentration ofrapamycin in the vitreous of the rabbit eye of at least 0.01 ng/mL forat least 30, at least 60, at least 90, or at least 120 days afteradministration of the liquid rapamycin formulation to the rabbit eyes.In some variations, the liquid rapamycin formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers rapamycingiving an average concentration of rapamycin in the vitreous of therabbit eye of at least 0.1 ng/mL for at least 30, at least 60, at least90, or at least 120 days after administration of the liquid rapamycinformulation to the rabbit eyes. In some variations, the liquid rapamycinformulation when injected between the sclera and conjunctiva of a rabbiteye delivers rapamycin giving an average concentration of rapamycin inthe vitreous of the rabbit eye of at least 0.5 ng/mL for at least 30, atleast 60, at least 90, or at least 120 days after administration of theliquid rapamycin formulation to the rabbit eyes.

In some variations, the liquid rapamycin formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers rapamycingiving an average concentration of rapamycin in the vitreous of therabbit eye of between 0.001 ng/mL and 10.0 ng/mL for at least 30, atleast 60, at least 90, or at least 120 days after administration of theliquid rapamycin formulation to the rabbit eyes. In some variations, theliquid rapamycin formulation when injected between the sclera andconjunctiva of a rabbit eye delivers rapamycin giving an averageconcentration of rapamycin in the vitreous of the rabbit eye of between0.01 ng/mL and 10 ng/mL for at least 30, at least 60, at least 90, or atleast 120 days after administration of the liquid rapamycin formulationto the rabbit eyes. In some variations, the liquid rapamycin formulationwhen injected between the sclera and conjunctiva of a rabbit eyedelivers rapamycin giving an average concentration of rapamycin in thevitreous of the rabbit eye of between 0.1 ng/mL and 10 ng/mL for atleast 30, at least 60, at least 90, or at least 120 days afteradministration of the liquid rapamycin formulation to the rabbit eyes.

In some variations, the liquid rapamycin formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers rapamycingiving an average concentration of rapamycin in the vitreous of therabbit eye of between 0.5 ng/mL and 10.0 ng/mL for at least 30, at least60, at least 90, or at least 120 days after administration of the liquidrapamycin formulation to the rabbit eyes.

In some variations, the liquid rapamycin formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers rapamycingiving a ratio of a maximum average concentration of rapamycin in thevitreous of a rabbit eye to a minimum average concentration of rapamycinin the vitreous of a rabbit eye less than 100 for days 30 to at least60, at least 90, or at least 120 days after administration of the liquidrapamycin formulation to the rabbit eyes. In some variations, the liquidrapamycin formulation when injected between the sclera and conjunctivaof a rabbit eye delivers rapamycin giving a ratio of a maximum averageconcentration of rapamycin in the vitreous of a rabbit eye to a minimumaverage concentration of rapamycin in the vitreous of a rabbit eye lessthan 50 for days 30 to at least 60, at least 90, or at least 120 daysafter administration of the liquid rapamycin formulation to the rabbiteyes. In some variations, the liquid rapamycin formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers rapamycingiving a ratio of a maximum average concentration of rapamycin in thevitreous of a rabbit eye to a minimum average concentration of rapamycinin the vitreous of a rabbit eye less than 10 for days 30 to at least 60,at least 90, or at least 120 days after administration of the liquidrapamycin formulation to the rabbit eyes. In he onset of, or causeregression of CNV, and thus may be used to treat, prevent, inhibit,delay the t, delay the onset of, or cause regression of wet AMD. It isbelieved that by changing certain concentration of rapamycin in thevitreous of a rabbit eye to a minimum average concentration of rapamycinin the vitreous of a rabbit eye less than 5 for days 30 to at least 60,at least 90, or at least 120 days after administration of the liquidrapamycin formulation to the rabbit eyes.

In some variations, the liquid rapamycin formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers rapamycingiving an average concentration of rapamycin in the vitreous of a rabbiteye that is approximately constant at a value greater than 0.001 ng/mLfor days 30 to at least 60, at least 90, or at least 120 days afteradministration of the solution to the rabbit eyes. In some variations,the liquid rapamycin formulation when injected between the sclera andconjunctiva of a rabbit eye delivers rapamycin giving an averageconcentration of rapamycin in the vitreous of a rabbit eye that isapproximately constant at a value greater than 0.01 ng/mL for days 30 toat least 60, at least 90, or at least 120 days after administration ofthe liquid rapamycin formulation to the rabbit eyes. In some variations,the liquid rapamycin formulation when injected between the sclera andconjunctiva of a rabbit eye delivers rapamycin giving an averageconcentration of rapamycin in the vitreous of a rabbit eye that isapproximately constant at a value greater than 0.1 ng/mL for days 30 toat least 60, at least 90, or at least 120 days after administration ofthe liquid rapamycin formulation to the rabbit eyes. In some variations,the liquid rapamycin formulation when injected between the sclera andconjunctiva of a rabbit eye delivers rapamycin giving an averageconcentration of rapamycin in the vitreous of a rabbit eye that isapproximately constant at a value of 1.0 ng/mL for days 30 to at least60, at least 90, or at least 120 days after administration of the liquidrapamycin formulation to the rabbit eyes.

In some variations, the liquid rapamycin formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers rapamycingiving an average concentration of rapamycin in the retina choroidtissues of the rabbit eye of at least 0.001 ng/mg for at least 30, atleast 60, at least 90, or at least 120 days after administration of theliquid rapamycin formulation to the rabbit eyes. In some variations, theliquid rapamycin formulation when injected between the sclera andconjunctiva of a rabbit eye delivers rapamycin giving an averageconcentration of rapamycin in the retina choroid tissues of the rabbiteye of at least 0.005 ng/mg for at least 30, at least 60, at least 90,or at least 120 days after administration of the liquid rapamycinformulation to the rabbit eyes. In some variations, the liquid rapamycinformulation when injected between the sclera and conjunctiva of a rabbiteye delivers rapamycin giving an average concentration of rapamycin inthe retina choroid tissues of the rabbit eye of at least 0.01 ng/mg forat least 30, at least 60, at least 90, or at least 120 days afteradministration of the liquid rapamycin formulation to the rabbit eyes.

In some variations, the liquid rapamycin formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers rapamycingiving an average concentration of rapamycin in the retina choroidtissues of the rabbit eye of between 0.001 ng/mg and 1.0 ng/mg for atleast 30, at least 60, at least 90, or at least 120 days afteradministration of the liquid rapamycin formulation to the rabbit eyes.In some variations, the liquid rapamycin formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers rapamycingiving an average concentration of rapamycin in the retina choroidtissues of the rabbit eye of between 0.001 ng/mg and 0.50 ng/mg for atleast 30, at least 60, at least 90, or at least 120 days afteradministration of the liquid rapamycin formulation to the rabbit eyes.In some variations, the liquid rapamycin formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers rapamycingiving an average concentration of rapamycin in the retina choroidtissues of the rabbit eye of between 0.001 ng/mg and 0.15 ng/mg for atleast 30, at least 60, at least 90, or at least 120 days afteradministration of the liquid rapamycin formulation to the rabbit eyes.In some variations, the liquid rapamycin formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers rapamycingiving an average concentration of rapamycin in the retina choroidtissues of the rabbit eye of between 0.001 ng/mg and 0.1 ng/mg for atleast 30, at least 60, at least 90, or at least 120 days afteradministration of the liquid rapamycin formulation to the rabbit eyes.

In some variations, the liquid rapamycin formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers rapamycingiving an average concentration of rapamycin in the retina choroidtissues of the rabbit eye of between 0.005 ng/mg and 1.0 ng/mg for atleast 30, at least 60, at least 90, or at least 120 days afteradministration of the liquid rapamycin formulation to the rabbit eyes.In some variations, the liquid rapamycin formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers rapamycingiving an average concentration of rapamycin in the retina choroidtissues of the rabbit eye of between 0.005 ng/mg and 0.50 ng/mg for atleast 30, at least 60, at least 90, or at least 120 days afteradministration of the liquid rapamycin formulation to the rabbit eyes.In some variations, the liquid rapamycin formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers rapamycingiving an average concentration of rapamycin in the retina choroidtissues of the rabbit eye of between 0.005 ng/mg and 0.15 ng/mg for atleast 30, at least 60, at least 90, or at least 120 days afteradministration of the liquid rapamycin formulation to the rabbit eyes.In some variations, the liquid rapamycin formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers rapamycingiving an average concentration of rapamycin in the retina choroidtissues of the rabbit eye of between 0.005 ng/mg and 0.1 ng/mg for atleast 30, at least 60, at least 90, or at least 120 days afteradministration of the liquid rapamycin formulation to the rabbit eyes.

In some variations, the liquid rapamycin formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers rapamycingiving an average concentration of rapamycin in the retina choroidtissues of the rabbit eye of between 0.01 ng/mg and 1.0 ng/mg for atleast 30, at least 60, at least 90, or at least 120 days afteradministration of the liquid rapamycin formulation to the rabbit eyes.In some variations, the liquid rapamycin formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers rapamycingiving an average concentration of rapamycin in the retina choroidtissues of the rabbit eye of between 0.01 ng/mg and 0.50 ng/mg for atleast 30, at least 60, at least 90, or at least 120 days afteradministration of the liquid rapamycin formulation to the rabbit eyes.In some variations, the liquid rapamycin formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers rapamycingiving an average concentration of rapamycin in the retina choroidtissues of the rabbit eye of between 0.01 ng/mg and 0.15 ng/mg for atleast 30, at least 60, at least 90, or at least 120 days afteradministration of the liquid rapamycin formulation to the rabbit eyes.In some variations, the liquid rapamycin formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers rapamycingiving an average concentration of rapamycin in the retina choroidtissues of the rabbit eye of between 0.01 ng/mg and 0.1 ng/mg for atleast 30, at least 60, at least 90, or at least 120 days afteradministration of the liquid rapamycin formulation to the rabbit eyes.

In some variations, the liquid rapamycin formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers rapamycingiving a ratio of a maximum average concentration of rapamycin in theretina choroid tissues of a rabbit eye to a minimum averageconcentration of rapamycin in the retina choroid tissues of a rabbit eyeless than 100 for days 30 to at least 60, at least 90, or at least 120days after administration of the liquid rapamycin formulation to therabbit eyes. In some variations, the liquid rapamycin formulation wheninjected between the sclera and conjunctiva of a rabbit eye deliversrapamycin giving a ratio of a maximum average concentration of rapamycinin the retina choroid tissues of a rabbit eye to a minimum averageconcentration of rapamycin in the retina choroid tissues of a rabbit eyeless than 50 for days 30 to at least 60, at least 90, or at least 120days after administration of the liquid rapamycin formulation to therabbit eyes. In some variations, the liquid rapamycin formulation wheninjected between the sclera and conjunctiva of a rabbit eye deliversrapamycin giving a ratio of a maximum average concentration of rapamycinin the retina choroid tissues of a rabbit eye to a minimum averageconcentration of rapamycin in the retina choroid tissues of a rabbit eyeless than 10 for days 30 to at least 60, at least 90, or at least 120days after administration of the liquid rapamycin formulation to therabbit eyes. In some variations, the liquid rapamycin formulation wheninjected between the sclera and conjunctiva of a rabbit eye deliversrapamycin giving a ratio of a maximum average concentration of rapamycinin the retina choroid tissues of a rabbit eye to a minimum averageconcentration of rapamycin in the retina choroid tissues of a rabbit eyeless than 5 for days 30 to at least 60, at least 90, or at least 120days after administration of the liquid rapamycin formulation to therabbit eyes.

In some variations, the liquid rapamycin formulation when injectedbetween the sclera and conjunctiva of a rabbit eye delivers rapamycingiving an average concentration of rapamycin in the retina choroidtissues of a rabbit eye that is approximately constant at a valuegreater than 0.001 ng/mg for days 30 to at least 60, at least 90, or atleast 120 days after administration of the liquid rapamycin formulationto the rabbit eyes. In some variations, the liquid rapamycin formulationwhen injected between the sclera and conjunctiva of a rabbit eyedelivers rapamycin giving an average concentration of rapamycin in theretina choroid tissues of a rabbit eye that is approximately constant ata value greater than 0.005 ng/mg for days 30 to at least 60, at least90, or at least 120 days after administration of the liquid rapamycinformulation to the rabbit eyes. In some variations, the liquid rapamycinformulation when injected between the sclera and conjunctiva of a rabbiteye delivers rapamycin giving an average concentration of rapamycin inthe retina choroid tissues of a rabbit eye that is approximatelyconstant at a value greater than 0.01 ng/mg for days 30 to at least 60,at least 90, or at least 120 days after administration of the liquidrapamycin formulation to the rabbit eyes.

In some variations, the liquid rapamycin formulation when injected intothe vitreous of a rabbit eye delivers rapamycin giving an averageconcentration of rapamycin in the vitreous of the rabbit eye of at least100 ng/mL for at least 30, at least 60, at least 90, or at least 120days after administration of the liquid rapamycin formulation to therabbit eyes. In some variations, the liquid rapamycin formulation wheninjected into the vitreous of a rabbit eye delivers rapamycin giving anaverage concentration of rapamycin in the vitreous of the rabbit eye ofat least 1000 ng/mL for at least 30, at least 60, at least 90, or atleast 120 days after administration of the liquid rapamycin formulationto the rabbit eyes. In some variations, the liquid rapamycin formulationwhen injected into the vitreous of a rabbit eye delivers rapamycingiving an average concentration of rapamycin in the vitreous of therabbit eye of at least 10,000 ng/mL for at least 30, at least 60, atleast 90, or at least 120 days after administration of the liquidrapamycin formulation to the rabbit eyes.

In some variations, the liquid rapamycin formulation when injected intothe vitreous of a rabbit eye delivers rapamycin giving an averageconcentration of rapamycin in the vitreous of the rabbit eye between 100ng/mL and 100,000 ng/mL for day 30 to at least 60, at least 90, or atleast 120 days after administration of the liquid rapamycin formulationto the rabbit eyes. In some variations, the liquid rapamycin formulationwhen injected into the vitreous of a rabbit eye delivers rapamycingiving an average concentration of rapamycin in the vitreous of therabbit eye between 100 ng/mL and 50,000 ng/mL for day 30 to at least 60,at least 90, or at least 120 days after administration of the liquidrapamycin formulation to the rabbit eyes.

In some variations, the liquid rapamycin formulation when injected intothe vitreous of a rabbit eye delivers rapamycin giving an averageconcentration of rapamycin in the vitreous of the rabbit eye between1000 ng/mL and 100,000 ng/mL for day 30 to at least 60, at least 90, orat least 120 days after administration of the liquid rapamycinformulation to the rabbit eyes. In some variations, the liquid rapamycinformulation when injected into the vitreous of a rabbit eye deliversrapamycin giving an average concentration of rapamycin in the vitreousof the rabbit eye between 1000 ng/mL and 50,000 ng/mL for day 30 to atleast 60, at least 90, or at least 120 days after administration of theliquid rapamycin formulation to the rabbit eyes.

In some variations, the liquid rapamycin formulation when injected intothe vitreous of a rabbit eye delivers rapamycin giving a ratio of amaximum average concentration of rapamycin in the vitreous of the rabbiteye to a minimum average concentration of rapamycin in the vitreous ofthe rabbit eye less than 100 for days 30 to at least 60, at least 90, orat least 120 days after administration of the liquid rapamycinformulation to the rabbit eyes. In some variations, the liquid rapamycinformulation when injected into the vitreous of a rabbit eye deliversrapamycin giving a ratio of a maximum average concentration of rapamycinin the vitreous of the rabbit eye to a minimum average concentration ofrapamycin in the vitreous of the rabbit eye less than 50 for days 30 toat least 60, at least 90, or at least 120 days after administration ofthe liquid rapamycin formulation to the rabbit eyes. In some variations,the liquid rapamycin formulation when injected into the vitreous of arabbit eye delivers rapamycin giving a ratio of a maximum averageconcentration of rapamycin in the vitreous of the rabbit eye to aminimum average concentration of rapamycin in the vitreous of the rabbiteye less than 10 for days 30 to at least 60, at least 90, or at least120 days after administration of the liquid rapamycin formulation to therabbit eyes.

In some variations, the liquid rapamycin formulation when injected intothe vitreous of a rabbit eye delivers rapamycin giving an averageconcentration of rapamycin in the vitreous of the rabbit eye that isapproximately constant at a value greater than 100 ng/mL for days 30 toat least 60, at least 90, or at least 120 days after administration ofthe liquid rapamycin formulation to the rabbit eyes. In some variations,the liquid rapamycin formulation when injected into the vitreous of arabbit eye delivers rapamycin giving an average concentration ofrapamycin in the vitreous of the rabbit eye that is approximatelyconstant at a value greater than 1000 ng/mL for days 30 to at least 60,at least 90, or at least 120 days after administration of the liquidrapamycin formulation to the rabbit eyes. In some variations, the liquidrapamycin formulation when injected into the vitreous of a rabbit eyedelivers rapamycin giving an average concentration of rapamycin in thevitreous of the rabbit eye that is approximately constant at a valuegreater than 10,000 ng/mL for days 30 to at least 60, at least 90, or atleast 120 days after administration of the liquid rapamycin formulationto the rabbit eyes.

In some variations, the liquid rapamycin formulation when injected intothe vitreous of a rabbit eye delivers rapamycin giving an averageconcentration of rapamycin in the retina choroid tissues of the rabbiteye of at least 0.001 ng/mg for at least 30, at least 60, at least 90,or at least 120 days after administration of the liquid rapamycinformulation to the rabbit eyes. In some variations, the liquid rapamycinformulation when injected into the vitreous of a rabbit eye deliversrapamycin giving an average concentration of rapamycin in the retinachoroid tissues of the rabbit eye of at least 0.01 ng/mg for at least30, at least 60, at least 90, or at least 120 days after administrationof the liquid rapamycin formulation to the rabbit eyes. In somevariations, the liquid rapamycin formulation when injected into thevitreous of a rabbit eye delivers rapamycin giving an averageconcentration of rapamycin in the retina choroid tissues of the rabbiteye of at least 0.05 ng/mg for at least 30, at least 60, at least 90, orat least 120 days after administration of the liquid rapamycinformulation to the rabbit eyes. In some variations, the liquid rapamycinformulation when injected into the vitreous of a rabbit eye deliversrapamycin giving an average concentration of rapamycin in the retinachoroid tissues of the rabbit eye of at least 0.10 ng/mg for at least30, at least 60, at least 90, or at least 120 days after administrationof the liquid rapamycin formulation to the rabbit eyes.

In some variations, the liquid rapamycin formulation when injected intothe vitreous of a rabbit eye delivers rapamycin giving an averageconcentration of rapamycin in the retina choroid tissues of the rabbiteye between 0.001 ng/mg and 10.00 ng/mg for at least 30, at least 60, atleast 90, or at least 120 days after administration of the liquidrapamycin formulation to the rabbit eyes. In some variations, the liquidrapamycin formulation when injected into the vitreous of a rabbit eyedelivers rapamycin giving an average concentration of rapamycin in theretina choroid tissues of the rabbit eye between 0.001 ng/mg and 5.00ng/mg for at least 30, at least 60, at least 90, or at least 120 daysafter administration of the liquid rapamycin formulation to the rabbiteyes. In some variations, the liquid rapamycin formulation when injectedinto the vitreous of a rabbit eye delivers rapamycin giving an averageconcentration of rapamycin in the retina choroid tissues of the rabbiteye between 0.001 ng/mg and 1.00 ng/mg for at least 30, at least 60, atleast 90, or at least 120 days after administration of the liquidrapamycin formulation to the rabbit eyes.

In some variations, the liquid rapamycin formulation when injected intothe vitreous of a rabbit eye delivers rapamycin giving an averageconcentration of rapamycin in the retina choroid tissues of the rabbiteye between 0.01 ng/mg and 10.00 ng/mg for at least 30, at least 60, atleast 90, or at least 120 days after administration of the liquidrapamycin formulation to the rabbit eyes. In some variations, the liquidrapamycin formulation when injected into the vitreous of a rabbit eyedelivers rapamycin giving an average concentration of rapamycin in theretina choroid tissues of the rabbit eye between 0.01 ng/mg and 5.00ng/mg for at least 30, at least 60, at least 90, or at least 120 daysafter administration of the liquid rapamycin formulation to the rabbiteyes. In some variations, the liquid rapamycin formulation when injectedinto the vitreous of a rabbit eye delivers rapamycin giving an averageconcentration of rapamycin in the retina choroid tissues of the rabbiteye between 0.01 ng/mg and 1.00 ng/mg for at least 30, at least 60, atleast 90, or at least 120 days after administration of the liquidrapamycin formulation to the rabbit eyes.

In some variations, the liquid rapamycin formulation when injected intothe vitreous of a rabbit eye delivers rapamycin giving an averageconcentration of rapamycin in the retina choroid tissues of the rabbiteye between 0.05 ng/mg and 10.00 ng/mg for at least 30, at least 60, atleast 90, or at least 120 days after administration of the liquidrapamycin formulation to the rabbit eyes. In some variations, the liquidrapamycin formulation when injected into the vitreous of a rabbit eyedelivers rapamycin giving an average concentration of rapamycin in theretina choroid tissues of the rabbit eye between 0.05 ng/mg and 5.00ng/mg for at least 30, at least 60, at least 90, or at least 120 daysafter administration of the liquid rapamycin formulation to the rabbiteyes. In some variations, the liquid rapamycin formulation when injectedinto the vitreous of a rabbit eye delivers rapamycin giving an averageconcentration of rapamycin in the retina choroid tissues of the rabbiteye between 0.05 ng/mg and 1.00 ng/mg for at least 30, at least 60, atleast 90, or at least 120 days after administration of the liquidrapamycin formulation to the rabbit eyes.

In some variations, the liquid rapamycin formulation when injected intothe vitreous of a rabbit eye delivers rapamycin giving an averageconcentration of rapamycin in the retina choroid tissues of the rabbiteye between 0.10 ng/mg and 10.00 ng/mg for at least 30, at least 60, atleast 90, or at least 120 days after administration of the liquidrapamycin formulation to the rabbit eyes. In some variations, the liquidrapamycin formulation when injected into the vitreous of a rabbit eyedelivers rapamycin giving an average concentration of rapamycin in theretina choroid tissues of the rabbit eye between 0.10 ng/mg and 5.00ng/mg for at least 30, at least 60, at least 90, or at least 120 daysafter administration of the liquid rapamycin formulation to the rabbiteyes. In some variations, the liquid rapamycin formulation when injectedinto the vitreous of a rabbit eye delivers rapamycin giving an averageconcentration of rapamycin in the retina choroid tissues of the rabbiteye between 0.10 ng/mg and 1.00 ng/mg for at least 30, at least 60, atleast 90, or at least 120 days after administration of the liquidrapamycin formulation to the rabbit eyes.

In some variations, the liquid rapamycin formulation when injected intothe vitreous of a rabbit eye delivers rapamycin giving a ratio of amaximum average concentration of rapamycin in the retina choroid tissuesof the rabbit eye to a minimum average concentration of rapamycin in theretina choroid tissues of the rabbit eye less than 100 for days 30 to atleast 60, at least 90, or at least 120 days after administration of theliquid rapamycin formulation to the rabbit eyes. In some variations, theliquid rapamycin formulation when injected into the vitreous of a rabbiteye delivers rapamycin giving a ratio of a maximum average concentrationof rapamycin in the retina choroid tissues of the rabbit eye to aminimum average concentration of rapamycin in the retina choroid tissuesof the rabbit eye less than 50 for days 30 to at least 60, at least 90,or at least 120 days after administration of the liquid rapamycinformulation to the rabbit eyes.

In some variations, the ratio of the base ten logarithms of the averagelevels of rapamycin in the vitreous and the retina choroid tissues isapproximately constant over an extended period of time. Put another way,as the level of rapamycin in the vitreous rises, the level of rapamycinin the retina choroid tissues rises to a similar degree when consideredon the logarithmic scale, and vice versa.

In some variations, the ratio of the base ten logarithms of the averagelevels of rapamycin in the vitreous versus the retina choroid tissues isapproximately constant over an extended period of time of 7, 30, 60, or90 days.

For treatment, prevention, inhibition, delaying the onset of, or causingthe regression of certain diseases or conditions, it may be desirable tomaintain delivery of a therapeutically effective amount of rapamycin foran extended period of time. Depending on the disease or condition beingtreated, prevented, inhibited, having onset delayed, or being caused toregress this extended period of time may be at least about 1 week, atleast about 2 weeks, at least about 3 weeks, at least about 1 month, atleast about 3 months, at least about 6 months, at least about 9 months,or at least about 1 year. Generally, however, any extended period ofdelivery may be possible. A therapeutically effective amount of agentmay be delivered for an extended period by a liquid formulation orcomposition that maintains for the extended period a concentration ofagent in a subject or an eye of a subject sufficient to deliver atherapeutically effective amount of agent for the extended time.

Delivery of a therapeutically effective amount of rapamycin for anextended period may be achieved via a single administration of a liquidrapamycin formulation or may be achieved by administration of two ormore doses of a liquid rapamycin formulation. As a non-limiting exampleof such multiple applications, maintenance of the therapeutic amount ofrapamycin for 3 months for treatment, prevention, inhibition, delay ofonset, or cause of regression of wet AMD may be achieved byadministration of one dose of a liquid rapamycin formulation deliveringa therapeutic amount for 3 months or by sequential application of aplurality of doses of a liquid rapamycin formulation. The optimal dosageregime will depend on the therapeutic amount of rapamycin needing to bedelivered, the period over which it need be delivered, and the deliverykinetics of the liquid formulation. Those versed in such extendedtherapeutic agent delivery dosing will understand how to identify dosingregimes that may be used based on the teachings described herein.

When using rapamycin for the treatment, prevention, inhibition, delayingthe onset of, or causing the regression of certain diseases, it may bedesirable for delivery of the rapamycin not to commence immediately uponplacement of the liquid formulation or composition into the eye region,but for delivery to commence after some delay. For example, but in noway limiting, such delayed release may be useful where the rapamycininhibits or delays wound healing and delayed release is desirable toallow healing of any wounds occurring upon placement of the liquidformulation or composition. Depending on the therapeutic agent beingdelivered and/or the diseases and conditions being treated, prevented,inhibited, onset delayed, and regression caused this period of delaybefore delivery of rapamycin commences may be about 1 hour, about 6hours, about 12 hours, about 18 hours, about 1 day, about 2 days, about3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8days, about 9 days, about 10 days, about 11 days, about 12 days, about13 days, about 14 days, about 21 days, about 28 days, about 35 days, orabout 42 days. Other delay periods may be possible. Delayed releaseformulations that may be used are known to people versed in thetechnology.

Routes of Administration

“Retina choroid” and “retina choroid tissues,” as used herein, aresynonymous and refer to the combined retina and choroid tissues of theeye.

“Subconjunctival” placement or injection, as used herein, refers toplacement or injection, respectively, between the sclera andconjunctiva. Subconjunctival is sometimes referred to herein as“sub-conj” administration.

By way of nonlimiting example, the liquid rapamycin formulationdescribed herein may be administered to the vitreous, aqueous humor,sclera, conjunctiva, between the sclera and conjunctiva, the retinachoroid tissues, macula, or other area in or proximate to the eye of ahuman subject, in amounts and for a duration effective to treat,prevent, inhibit, delay the onset of, or cause the regression of CNV andwet AMD.

Periocular routes of delivery may deliver rapamycin to the retinawithout some of the risks of intravitreal delivery. Periocular routesinclude but are not limited to subconjunctival, subtenon, retrobulbar,peribulbar and posterior juxtascleral delivery. A “periocular” route ofadministration means placement near or around the eye. For a descriptionof exemplary periocular routes for retinal drug delivery, see Periocularroutes for retinal drug delivery, Raghava et al. (2004), Expert Opin.Drug Deliv. 1(1):99-114, which is incorporated herein by reference inits entirety.

In some variations the liquid formulations described herein areadministered intraocularly. Intraocular administration includesplacement or injection within the eye, including in the vitreous.

In some variations, an effective amount of rapamycin is placedintravitreally or subconjunctivally to treat, prevent, inhibit, delaythe onset of, or cause the regression of CNV, wet AMD, or dry AMD.

Intravitreal administration is more invasive than some other types ofocular procedures. Because of the potential risks of adverse effects,intravitreal administration may not be optimal for treatment ofrelatively healthy eyes. By contrast, periocular administration, such assubconjunctival administration, is much less invasive than intravitrealadministration. When rapamycin is delivered by a periocular route, itmay be possible to treat patients with healthier eyes than could betreated using intravitreal administration. In some variations,subconjunctival injection is used to prevent or delay onset of a diseaseor condition of the eye, where the eye of the subject has visual acuityof 20/40 or better.

Routes of administration that may be used to administer a liquidformulation include but are not limited to placement of the liquidformulation, for example by injection, into an aqueous medium in thesubject, including but not limited to subconjunctival and intravitrealplacement, including but not limited to injection.

Compositions and liquid formulations comprising rapamycin can beadministered directly to the eye using a variety of procedures,including but not limited to procedures in which (1) rapamycin isadministered by injection, including but not limited to administrationby using a syringe and hypodermic needle, an insulin needle, or aHamilton HPLC-type needle, or (2) a specially designed device is used toinject rapamycin.

Intravitreal and Subconjunctival Delivery of Rapamycin for Treatment,Prevention, Inhibition, Delay of Onset, or Cause of Regression of AMD

As used herein, to “prevent” a disease or condition by administration ofrapamycin means that the detectable physical characteristics or symptomof the disease or condition do not develop following administration ofrapamycin.

As used herein, to “delay onset of” a disease or condition byadministration of rapamycin means that at least one detectable physicalcharacteristic or symptom of the disease or condition develops later intime following administration of rapamycin as compared to the progressof the disease or condition without administration of rapamycin.

As used herein, to “treat” a disease or condition by administration ofrapamycin means that the progress of at least one detectable physicalcharacteristic or symptom of the disease or condition is slowed,stopped, or reversed following administration of rapamycin as comparedto the progress of the disease or condition without administration ofrapamycin.

A subject having a predisposition for or in need of prevention may beidentified by the skilled practitioner by established methods andcriteria in the field given the teachings herein. The skilledpractitioner may also readily diagnose individuals as in need ofinhibition or treatment based upon established criteria in the field foridentifying angiogenesis and/or neovascularization given the teachingsherein.

As used herein, a “subject” is generally any animal that may benefitfrom administration of rapamycin as described herein. The rapamycin maybe administered to a mammal subject. Unless the context appearsotherwise, all of the methods described herein may be performed on ahuman subject. The rapamycin may be administered to a veterinary animalsubject. The rapamycin may be administered to a model experimentalanimal subject.

In some variations described herein, a solution comprising rapamycin isdelivered subconjunctivally or to the vitreous of an eye of a subject,including but not limited to a human subject, to prevent, treat,inhibit, delay onset of, or cause regression of angiogenesis in the eye,including but not limited to treating CNV as observed, for example, inAMD. In some variations, the solution is used to treat angiogenesis inthe eye, including but not limited to treating CNV as observed, forexample, in AMD. Rapamycin has been shown to inhibit CNV in rat and micemodels, as described in U.S. application Ser. No. 10/665,203, which isincorporated herein by reference in its entirety. Rapamycin has beenobserved to inhibit MATRIGEL and laser-induced CNV when administeredsystemically and subretinally.

In some variations, the formulations and pharmaceutical formulationsdescribed herein are used to prevent or delay onset of a disease orcondition of the eye where the subject, including but not limited to ahuman subject, is at heightened risk of developing the disease orcondition of the eye. A subject with a heightened risk of developing adisease or condition is a subject with one or more indications that thedisease or condition is likely to develop in the particular subject. Insome variations the subject with a heightened risk of developing wet AMDis a subject with dry AMD in at least one eye. In some variations thesubject with a heightened risk of developing wet AMD in a fellow eye isa subject with wet AMD in the other eye. In some variations, theformulations and pharmaceutical formulations described herein are usedto prevent or delay onset of CNV in a subject at heightened risk ofdeveloping CNV, including but not limited to prevention or delayingonset of CNV in the fellow eye of a subject, including but not limitedto a human subject with AMD in one eye. In some variations, theformulations and pharmaceutical formulations described herein are usedto prevent or delay onset of CNV in the fellow eye of a subject with wetAMD in one eye. In some variations, the formulations and pharmaceuticalformulations comprise a limus compound, including but not limited torapamycin. In some variations the formulations and pharmaceuticalformulations are administered subconjunctivally to an eye with vision of20/40 or better.

As described herein, the dosage of rapamycin will depend on thecondition being addressed, whether the condition is to be treated,prevented, inhibited, have onset delayed, or be caused to regress, theparticular therapeutic agent, and other clinical factors such as weightand condition of the subject and the route of administration of thetherapeutic agent. It is to be understood that the methods, liquidformulations, and compositions described herein have application forboth human and veterinary use, as well as uses in other possibleanimals. As described herein, tissue concentrations of rapamycinexpressed in units of mass per volume generally refer to tissues thatare primarily aqueous such as the vitreous, for example. Tissueconcentrations of rapamycin expressed in unit of mass per mass generallyrefer to other tissues such as the sclera or retina choroid tissues, forexample.

The liquid rapamycin formulations described herein may deliver rapamycinfor an extended period of time. One nonlimiting example of such anextended release delivery system is a liquid rapamycin formulation thatdelivers rapamycin to a subject or to the eye of a subject in an amountsufficient to treat, prevent, inhibit, delay onset of, or causeregression of wet age-related macular degeneration for an extendedperiod of time. In some variations, the liquid formulation is used totreat wet age-related macular degeneration for an extended period oftime. In some variations, the liquid formulation is used to prevent wetage-related macular degeneration for an extended period of time. In somevariations, the liquid formulation is used to prevent transition of dryAMD to wet AMD for an extended period of time.

One concentration of rapamycin that may be used in the methods describedherein is one that provides to a subject about 0.01 pg/ml or pg/mg ormore of rapamycin at the tissue level. Another concentration that may beused is one that provides to a subject about 0.1 pg/ml or ng/mg or moreat the tissue level. Another concentration that may be used is one thatprovides to a subject about 1 pg/ml or ng/mg or more at the tissuelevel. Another concentration that may be used is one that provides to asubject about 0.01 ng/ml or ng/mg or more at the tissue level. Anotherconcentration that may be used is one that provides to a subject about0.1 ng/ml or ng/mg or more at the tissue level. Another concentrationthat may be used is one that provides to a subject about 0.5 ng/ml orng/mg or more at the tissue level. Another concentration that may beused is one that provides to a subject about 1 ng/ml or more at thetissue level. Another concentration that may be used is one thatprovides to a subject about 2 ng/ml or more at the tissue level. Anotherconcentration that may be used is one that provides to a subject about 3ng/ml or more at the tissue level. Another concentration that may beused is one that provides to a subject about 5 ng/ml or more at thetissue level. Another concentration that may be used is one thatprovides to a subject about 10 ng/ml or more at the tissue level.Another concentration that may be used is one that provides to a subjectabout 15 ng/ml or more at the tissue level. Another concentration thatmay be used is one that provides to a subject about 20 ng/ml or more atthe tissue level. Another concentration that may be used is one thatprovides to a subject about 30 ng/ml or more at the tissue level.Another concentration that may be used is one that provides to a subjectabout 50 ng/ml or more at the tissue level. One of ordinary skill in theart would know how to arrive at an appropriate concentration dependingon the route and duration of administration utilized, given theteachings herein.

Generally, the amount of rapamycin administered in a liquid formulationis an amount sufficient to treat, prevent, inhibit, delay the onset, orcause regression of the disease or condition of the eye for the requiredamount of time. In some variations the amount of rapamycin administeredin the liquid formulation is an amount sufficient to treat the diseaseor condition of the eye for the required amount of time.

In some variations, a total amount of rapamycin less than about 5 mg isadministered subconjunctivally. In some variations, a total amount ofrapamycin less than about 5.0 mg is administered subconjunctivally. Insome variations, a total amount of rapamycin less than about 4.5 mg isadministered subconjunctivally. In some variations, a total amount ofrapamycin less than about 4.0 mg is administered subconjunctivally. Insome variations, a total amount of rapamycin less than about 3.5 mg isadministered subconjunctivally. In some variations, a total amount ofrapamycin less than about 3.0 mg is administered subconjunctivally. Insome variations, a total amount of rapamycin less than about 2.5 mg isadministered subconjunctivally. In some variations, a total amount ofrapamycin less than about 2 mg is administered subconjunctivally. Insome variations, a total amount of rapamycin less than about 1.2 mg isadministered subconjunctivally. In some variations, a total amount ofrapamycin less than about 1.0 mg is administered subconjunctivally. Insome variations, a total amount of rapamycin less than about 0.8 mg isadministered subconjunctivally. In some variations, a total amount ofrapamycin less than about 0.6 mg is administered subconjunctivally. Insome variations, a total amount of rapamycin less than about 0.4 mg isadministered subconjunctivally. In some variations, a volume of aformulation is administered that contains an amount of rapamycindescribed herein.

In some variations, a total amount of rapamycin less than about 200 μgis administered intravitreally. In some variations, a total amount ofrapamycin less than about 200 μg is administered intravitreally. In somevariations, a total amount of rapamycin less than about 300 μg isadministered intravitreally. In some variations, a total amount ofrapamycin less than about 400 μg is administered intravitreally. In somevariations, a total amount of rapamycin less than about 500 μg isadministered intravitreally. In some variations, a total amount ofrapamycin less than about 600 μg is administered intravitreally. In somevariations, a total amount of rapamycin less than about 800 μg isadministered intravitreally. In some variations, a total amount ofrapamycin less than about 1 mg is administered intravitreally. In somevariations, a total amount of rapamycin less than about 2 mg isadministered intravitreally. In some variations, a total amount ofrapamycin less than about 2.5 mg is administered intravitreally. In somevariations, a total amount of rapamycin less than about 3 mg isadministered intravitreally. In some variations, a total amount ofrapamycin less than about 3.5 mg is administered intravitreally. In somevariations, a total amount of rapamycin less than about 4 mg isadministered intravitreally. In some variations, a volume of aformulation is administered that contains an amount of rapamycindescribed herein.

In some variations, a liquid formulation containing a concentration ofrapamycin by weight of the total of between about 0.5% and about 6% issubconjunctivally administered to a human subject by administeringbetween about 0.1 μl and about 200 μl of a liquid formulation describedherein. In some variations, a liquid formulation containing aconcentration of rapamycin by weight of the total of between about 0.5%and about 4% is subconjunctivally administered to a human subject byadministering between about 1 μl and about 50 μl of a liquid formulationdescribed herein. In some variations, a liquid formulation containing aconcentration of rapamycin by weight of the total of between about 1.5%and about 3.5% is subconjunctivally administered to a human subject byadministering between about 1 μl and about 15 μl of a liquid formulationdescribed herein. In some variations, a liquid formulation containing aconcentration of rapamycin by weight of the total of about 2% issubconjunctivally administered to a human subject by administeringbetween about 1 μl and about 15 μl of a liquid formulation describedherein.

In some variations, a liquid formulation containing an amount ofrapamycin of between about 0.2 μg and about 4 mg is subconjunctivallyadministered to a human subject by administering between about 0.1 μland about 200 μl of a liquid formulation described herein. In somevariations, a liquid formulation containing an amount of rapamycin ofbetween about 20 μg and about 2 mg is subconjunctivally administered toa human subject by administering between about 1 μl and about 100 μl ofa liquid formulation described herein. In some variations, a liquidformulation containing an amount of rapamycin of between about 5 μg andabout 1 mg is subconjunctivally administered to a human subject byadministering between about 1 μl and about 50 μl of a liquid formulationdescribed herein. In some variations, a liquid formulation containing anamount of rapamycin of between about 20 μg and about 500 μg issubconjunctivally administered to a human subject by administeringbetween about 1 μl and about 25 μl of a liquid formulation describedherein. In some variations, a liquid formulation containing an amount ofrapamycin of between about 20 μg and about 300 μg is subconjunctivallyadministered to a human subject by administering between about 1 μl andabout 15 μl of a liquid formulation described herein.

In some variations, a total amount of rapamycin less than about 200 μgis administered intravitreally. In some variations, a total amount ofrapamycin less than about 200 μg is administered intravitreally. In somevariations, a total amount of rapamycin less than about 300 μg isadministered intravitreally. In some variations, a total amount ofrapamycin less than about 400 μg is administered intravitreally. In somevariations, a total amount of rapamycin less than about 500 μg isadministered intravitreally. In some variations, a total amount ofrapamycin less than about 600 μg is administered intravitreally. In somevariations, a total amount of rapamycin less than about 800 μg isadministered intravitreally. In some variations, a total amount ofrapamycin less than about 1 mg is administered intravitreally. In somevariations, a total amount of rapamycin less than about 2 mg isadministered intravitreally. In some variations, a total amount ofrapamycin less than about 2.5 mg is administered intravitreally. In somevariations, a total amount of rapamycin less than about 3 mg isadministered intravitreally. In some variations, a total amount ofrapamycin less than about 3.5 mg is administered intravitreally. In somevariations, a total amount of rapamycin less than about 4 mg isadministered intravitreally. In some variations, a volume of aformulation is administered that contains an amount of rapamycindescribed herein.

In some variations, a liquid formulation containing a concentration ofrapamycin by weight of the total of between about 0.5% and about 6% isintravitreally administered to a human subject by administering betweenabout 0.1 μl and about 200 μl of a liquid formulation described herein.In some variations, a liquid formulation containing a concentration ofrapamycin by weight of the total of between about 0.5% and about 4% isintravitreally administered to a human subject by administering betweenabout 1 μl and about 50 μl of a liquid formulation described herein. Insome variations, a liquid formulation containing a concentration ofrapamycin by weight of the total of between about 1.5% and about 3.5% isintravitreally administered to a human subject by administering betweenabout 1 μl and about 15 μl of a liquid formulation described herein. Insome variations, a liquid formulation containing a concentration ofrapamycin by weight of the total of about 2% is intravitreallyadministered to a human subject by administering between about 1 μl andabout 15 μl of a liquid formulation described herein.

In some variations, a liquid formulation containing an amount ofrapamycin of between about 0.2 μg and about 4 mg is intravitreallyadministered to a human subject by administering between about 0.1 μland about 200 μl of a liquid formulation described herein. In somevariations, a liquid formulation containing an amount of rapamycin ofbetween about 20 μg and about 2 mg is intravitreally administered to ahuman subject by administering between about 1 μl and about 100 μl of aliquid formulation described herein. In some variations, a liquidformulation containing an amount of rapamycin of between about 20 μg andabout 1 mg is intravitreally administered to a human subject byadministering between about 1 μl and about 50 μl of a liquid formulationdescribed herein. In some variations, a liquid formulation containing anamount of rapamycin of between about 20 μg and about 500 μg isintravitreally administered to a human subject by administering betweenabout 1 μl and about 25 μl of a liquid formulation described herein. Insome variations, a liquid formulation containing an amount of rapamycinof between about 20 μg and about 300 μg is intravitreally administeredto a human subject by administering between about 1 μl and about 15 μlof a liquid formulation described herein.

In some variations a formulation as described herein containing anamount of rapamycin of between about 1 μg and about 5 mg is administeredto a human subject for treatment of wet AMD. In some variations aformulation as described herein containing an amount of rapamycin ofbetween about 20 μg and about 4 mg is administered to a human subjectfor treatment of wet AMD. In some variations a liquid formulation asdescribed herein containing an amount of rapamycin of between about 20μg and about 1.2 mg is administered to a human subject for treatment ofwet AMD. In some variations an amount of rapamycin of between about 10μg and about 0.5 mg is administered to a human subject for treatment ofwet AMD. In some variations, an amount of rapamycin of between about 10μg and 90 μg is administered to a human subject for treatment of wetAMD. In some variations, an amount of rapamycin of between about 60 μgand 120 μg is administered to a human subject for treatment of wet AMD.In some variations, an amount of rapamycin of between about 100 μg and400 μg is administered to a human subject for treatment of wet AMD. Insome variations, an amount of rapamycin of between about 400 μg and 1 mgis administered to a human subject for treatment of wet AMD. In somevariations, an amount of rapamycin of between about 1 mg and 5 mg isadministered to a human subject for treatment of wet AMD. In somevariations, an amount of rapamycin of between about 3 mg and 7 mg isadministered to a human subject for treatment of wet AMD. In somevariations, an amount of rapamycin of between about 5 mg and 10 mg isadministered to a human subject for treatment of wet AMD.

In some variations a formulation as described herein containing anamount of rapamycin of between about 1 μg and about 5 mg is administeredto a human subject for prevention of wet AMD. In some variations aformulation as described herein containing an amount of rapamycin ofbetween about 20 μg and about 4 mg is administered to a human subjectfor prevention of wet AMD. In some variations a liquid formulation asdescribed herein containing an amount of rapamycin of between about 20μg and about 1.2 mg is administered to a human subject for prevention ofwet AMD. In some variations an amount of rapamycin of between about 10μg and about 0.5 mg is administered to a human subject for prevention ofwet AMD. In some variations, an amount of rapamycin of between about 10μg and 90 μg is administered to a human subject for prevention of wetAMD. In some variations, an amount of rapamycin of between about 60 μgand 120 μg is administered to a human subject for prevention of wet AMD.In some variations, an amount of rapamycin of between about 100 μg and400 μg is administered to a human subject for prevention of wet AMD. Insome variations, an amount of rapamycin of between about 400 μg and 1 mgis administered to a human subject for prevention of wet AMD. In somevariations, an amount of rapamycin of between about 1 mg and 5 mg isadministered to a human subject for prevention of wet AMD. In somevariations, an amount of rapamycin of between about 3 mg and 7 mg isadministered to a human subject for prevention of wet AMD. In somevariations, an amount of rapamycin of between about 5 mg and 10 mg isadministered to a human subject for prevention of wet AMD. In somevariations, prevention of wet AMD is prevention of the transition fromdry AMD to wet AMD.

In some variations a formulation as described herein containing anamount of rapamycin of between about 1 μg and about 5 mg is administeredto a human subject for treatment of dry AMD. In some variations aformulation as described herein containing an amount of rapamycin ofbetween about 20 μg and about 4 mg is administered to a human subjectfor treatment of dry AMD. In some variations a liquid formulation asdescribed herein containing an amount of rapamycin of between about 20μg and about 1.2 mg is administered to a human subject for treatment ofdry AMD. In some variations an amount of rapamycin of between about 10μg and about 0.5 mg is administered to a human subject for treatment ofdry AMD. In some variations, an amount of rapamycin of between about 10μg and 90 μg is administered to a human subject for treatment of dryAMD. In some variations, an amount of rapamycin of between about 60 μgand 120 μg is administered to a human subject for treatment of dry AMD.In some variations, an amount of rapamycin of between about 100 μg and400 μg is administered to a human subject for treatment of dry AMD. Insome variations, an amount of rapamycin of between about 400 μg and 1 mgis administered to a human subject for treatment of dry AMD. In somevariations, an amount of rapamycin of between about 1 mg and 5 mg isadministered to a human subject for treatment of dry AMD. In somevariations, an amount of rapamycin of between about 3 mg and 7 mg isadministered to a human subject for treatment of dry AMD. In somevariations, an amount of rapamycin of between about 5 mg and 10 mg isadministered to a human subject for treatment of dry AMD.

In some variations, a liquid formulation as described herein containingan amount of rapamycin of between about 1 μg and about 5 mg isadministered to a human subject for treatment of angiogenesis, includingbut not limited to choroidal neovascularization. In some variations fortreatment of angiogenesis, including but not limited to choroidalneovascularization, a formulation as described herein containing anamount of rapamycin of between about 1 μg and about 5 mg is administeredto a human subject. In some variations for treatment of angiogenesis,including but not limited to choroidal neovascularization, a formulationas described herein containing an amount of rapamycin of between about20 μg and about 4 mg, between about 20 μg and about 1.2 mg, betweenabout 10 μg and about 0.5 mg, between about 10 μg and 90 μg betweenabout 60 μg and 120 μg, between about 100 μg and 400 μg, between about400 μg and 1 mg, or between about 1 mg and 5 mg is administered to thehuman subject.

In some variations, any one or more of the rapamycin formulationsdescribed herein are administered intravitreally every 3 or more months,every 6 or more months, every 9 or more months, or every 12 or moremonths, or longer, to treat one or more of choroidal neovascularization,wet AMD, dry AMD, to prevent wet AMD, or to prevent progression of dryAMD to wet AMD. In some variations, any one or more of the rapamycinformulations described herein are administered subconjunctivally every 3or more months, every 6 or more months, every 9 or more months, or every12 or more months, or longer, to treat one or more of choroidalneovascularization, wet AMD, dry AMD, to prevent wet AMD, or to preventprogression of dry AMD to wet AMD. In some variations, the effect of therapamycin persists beyond the period during which it is present in theocular tissues.

In some variations, any one or more of the formulations described hereinare administered intravitreally every 3 or more months, every 6 or moremonths, every 9 or more months, or every 12 or more months, or longer,to prevent one or more of choroidal neovascularization, wet AMD, dryAMD, or to prevent wet AMD. In some variations, any one or more of theformulations described herein are administered subconjunctivally every 3or more months, every 6 or more months, every 9 or more months, or every12 or more months, or longer, to prevent one or more of choroidalneovascularization, wet AMD, dry AMD, or to prevent wet AMD.

Rapamycin may, for example, be delivered at a dosage range between about1 ng/day and about 100 μg/day, or at dosages higher or lower than thisrange, depending on the route and duration of administration. In somevariations of liquid formulation or composition used in the methodsdescribed herein, rapamycin is delivered at a dosage range of betweenabout 0.1 μg/day and about 10 μg/day. In some variations of liquidformulation or composition used in the methods described herein,rapamycin is delivered at a dosage range of between about 1 μg/day andabout 5 μg/day. Dosages of rapamycin for treatment, prevention,inhibition, delay of onset, or cause of regression of various diseasesand conditions described herein can be refined by the use of clinicaltrials.

The liquid formulations and compositions described herein may be usedfor delivery to the eye, as one nonlimiting example by ocular orperiocular administration, of therapeutically effective amounts ofrapamycin for extended periods of time to treat, prevent, inhibit, delaythe onset of, or cause regression of CNV, and thus may be used to treat,prevent, inhibit, delay the onset of, or cause regression of wet AMD. Itis believed that by changing certain characteristics of the liquidformulations and compositions described herein, including but notlimited to the volume, positioning and components of the liquidformulations, the liquid formulations and compositions described hereinmay be used to deliver therapeutically effective amounts of rapamycin tothe eye for a variety of extended time periods including delivery oftherapeutic amounts for greater than about 1 week, for greater thanabout 2 weeks, for greater than about 3 weeks, for greater than about 1month, for greater than about 3 months, for greater than about 6 months,for greater than about 9 months, for greater than about 1 year.

When a therapeutically effective amount of rapamycin is administered toa subject suffering from wet AMD, the rapamycin may treat, inhibit, orcause regression of the wet AMD. Different therapeutically effectiveamounts may be required for treatment, inhibition or causing regression.A subject suffering from wet AMD may have CNV lesions, and it isbelieved that administration of a therapeutically effective amount ofrapamycin may have a variety of effects, including but not limited tocausing regression of the CNV lesions, stabilizing the CNV lesion, andpreventing progression of an active CNV lesion.

When a therapeutically effective amount of rapamycin is administered toa subject suffering from dry AMD, it is believed that the rapamycin mayprevent or slow the progression of the dry AMD.

In some variations, a liquid rapamycin formulation described herein isadministered in combination with other therapeutic agents and therapies,including but not limited to agents and therapies useful for thetreatment, prevention, inhibition, delaying onset of, or causingregression of angiogenesis or neovascularization, particularly CNV. Insome variations the additional agent or therapy is used to treatregression of angiogenesis or neovascularization, particularly CNV.Non-limiting examples of such additional agents and therapies includepyrrolidine, dithiocarbamate (NFκB inhibitor); squalamine; TPN 470analogue and fumagillin; PKC (protein kinase C) inhibitors; Tie-1 andTie-2 kinase inhibitors; inhibitors of VEGF receptor kinase; proteosomeinhibitors such as VELCADE™ (bortezomib, for injection; ranibuzumab(LUCENTIS™) and other antibodies directed to the same target; pegaptanib(MACUGEN™); vitronectin receptor antagonists, such as cyclic peptideantagonists of vitronectin receptor-type integrins; α-v/β-3 integrinantagonists; α-v/β-1 integrin antagonists; thiazolidinediones such asrosiglitazone or troglitazone; interferon, including γ-interferon orinterferon targeted to CNV by use of dextran and metal coordination;pigment epithelium derived factor (PEDF); endostatin; angiostatin;tumistatin; canstatin; anecortave acetate; acetonide; triamcinolone;tetrathiomolybdate; RNA silencing or RNA interference (RNAi) ofangiogenic factors, including ribozymes that target VEGF expression;ACCUTANE™ (13-cis retinoic acid); ACE inhibitors, including but notlimited to quinopril, captopril, and perindozril; inhibitors of mTOR(mammalian target of rapamycin); 3-aminothalidomide; pentoxifylline;2-methoxyestradiol; colchicines; AMG-1470; cyclooxygenase inhibitorssuch as nepafenac, rofecoxib, diclofenac, rofecoxib, NS398, celecoxib,vioxx, and (E)-2-alkyl-2(4-methanesulfonylphenyl)-1-phenylethene; t-RNAsynthase modulator; metalloprotease 13 inhibitor; acetylcholinesteraseinhibitor; potassium channel blockers; endorepellin; purine analog of6-thioguanine; cyclic peroxide ANO-2; (recombinant) arginine deiminase;epigallocatechin-3-gallate; cerivastatin; analogues of suramin; VEGFtrap molecules; inhibitors of hepatocyte growth factor (antibodies tothe growth factor or its receptors, small molecular inhibitors of thec-met tyrosine kinase, truncated versions of HGF e.g. NK4); apoptosisinhibiting agents; VISUDYNE™, snET2 and other photo sensitizers withphotodynamic therapy (PDT); and laser photocoagulation.

EXAMPLES

Unless the context indicates otherwise, the error bars in the chartsshow one standard deviation. Where ethanol is used, it is 200 proofethanol from Gold Shield Distributors, Hayward, Calif. Where rapamycinis used, it is from LC laboratories, Woburn, Mass., or Chunghwa ChemicalSynthesis & Biotech Co., LTD (CCSB), Taipei Hsien, Taiwan, ROC. WherePEG 400 is used, it is from The Dow Chemical Company, New Milford, Conn.As used herein, “% w/w” means the weight of the component divided by thetotal formulation weight. Some of the graphs use the expression “uL” or“ug” to refer to μL or μg, respectively.

Example 1 Preparation and Characterization of a Rapamycin-ContainingSolution

About 320 g of ethanol was sparged with N₂ for about 10 minutes, andthen about 40 g of sirolimus was added to the ethanol. The mixture wassonicated for about 20 minutes, by the end of which all of the sirolimushad gone into solution to form a sirolimus stock solution. A diluentnon-aqueous liquid component was prepared by sonicating about 1880 g ofPEG 400 for about 60 minutes, and then sparging the non-aqueous liquidcomponent with nitrogen for about 10 minutes.

The sirolimus stock solution and the PEG 400 were then rotated at aboutroom temperature in a rotary evaporator for about 10 minutes to mix thestock solution with the diluent non-aqueous liquid component. Aftermixing, the solution was sparged with nitrogen for about 10 minutes andblanketed with nitrogen for about 5 minutes. After the solution wassparged and filled with nitrogen, about 240 g of excess ethanol wasevaporated from the solution by increasing the solution temperature,maintaining a temperature that did not exceed 40° C. for an extendedperiod of time and continuing to rotate the solution for about 2.5hours.

The resulting solution comprised about 40 g of sirolimus (about 2% w/w),about 80 g of ethanol (about 4% w/w), and about 1880 g of PEG 400 (about94% w/w). This solution was sparged with nitrogen for about 10 minutesand blanketed with nitrogen for about 5 minutes. The solution was thenfiltered through a 0.2 micron filter. HPLC vials were filled with 2 mleach of the filtered solution to leave a head space in each container ofabout 400 μl. This head space was filled with nitrogen gas and capped.

Example 2 Preparation and Characterization of a Rapamycin-ContainingSolution

Rapamycin, ethanol and PEG 400 were placed in a container to give finalconcentrations by weight of about 2.00% w/w rapamycin, about 4.00% w/wethanol, and about 94.00% w/w PEG 400. The mixture was capped andsonicated for 1-2 hours. The sonication generated heat, withtemperatures of up to about 40 or 50° C. Volumes of 1 μl, 3 μl, 20 μl,and 40 μl formed a non-dispersed mass in the vitreous of rabbit eyes.

Example 3 Subconjunctival Injection of a Rapamycin-Containing Solution

20 μl of the solution described in Example 2 were injected between thesclera and the conjunctiva of the eye of New Zealand white rabbits. FIG.2 depicts the level of rapamycin in the vitreous on a logarithmic scaleat 5, 30, 60, 90, and 120 days after injection. FIG. 3 depicts the levelof rapamycin in the retina choroid on a logarithmic scale at the sametime points. For comparison, FIG. 2 and FIG. 3 also depict results ofsimilar studies, performed with 40 μl and 60 injections, described belowin Example 4 and Example 5.

In FIGS. 2-5, discussed in this and following examples, some outlierpoints have been omitted. Individual data points from the same study atthe same time point were compared to each other. When the arithmeticmean of the data points was lower than their standard deviation, thedata points that were higher or lower by an order of magnitude wereconsidered as outliers.

The analysis was by liquid chromatography mass spectroscopy (LCMS) usingan internal standard.

At each time point, the average concentration of rapamycin wascalculated by adding the concentrations of rapamycin obtained for eacheye from each rabbit, and dividing the total by the number of eyesanalyzed.

The full vitreous was homogenized and analyzed. The averageconcentration of the vitreous was calculated by dividing the mass ofrapamycin measured by the volume of vitreous analyzed. Where injectionis intravitreal, for samples other than the vitreous, the sample did notinclude the site of administration; thus, this measurement indicated thelevel of rapamycin delivered to the vitreous via the solution. Whereinjection was intravitreal, for vitreous samples, the sample is thoughtto include the site of administration; thus, this measurement indicatedthe level of rapamycin cleared from the vitreous.

The full retina choroid was homogenized and analyzed. The averageconcentration of the retina choroid was calculated by dividing the massof rapamycin measured by the mass of retina choroid analyzed.

Where injection was intravitreal or subconjunctival, the sample did notinclude the site of administration; thus, this measurement indicated thelevel of rapamycin delivered to the retina choroid via the solution.

In this experiment, between two and five rabbit eyes were analyzed ateach time point. The vitreous sample did not include the site ofadministration, so this measurement indicated the level of rapamycindelivered to the vitreous. The average level of rapamycin in thevitreous at 5, 30, 60, 90, and 120 days after subconjunctival injectionwas about 1.81, 0.45, 0.39, 1.85, and 1.49 ng/ml, respectively.

The retina choroid was homogenized and analyzed as described in Example3, with the samples taken as described for the vitreous above. Theretina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 5, 30,60, 90, and 120 days after subconjunctival injection was about 0.14,0.03, 0.02, 0.02, and 0.01 ng/mg, respectively.

Example 4 Subconjunctival Injection of a Rapamycin-Containing Solution

40 μl of the solution described in Example 2 were injected between thesclera and the conjunctiva of the eye of New Zealand white rabbits. FIG.2 depicts the level of rapamycin in the vitreous on a logarithmic scaleat 5, 30, 60, 90, and 120 days after injection. FIG. 3 depicts the levelof rapamycin in the retina choroid on a logarithmic scale at the sametime points.

The vitreous was homogenized and analyzed as described in Example 3.Between two and five rabbit eyes were analyzed at each time point. Thevitreous sample did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the vitreous.The average level of rapamycin in the vitreous at 5, 30, 60, 90, and 120days after subconjunctival injection was about 2.39, 0.65, 0.54, 2.07,and 1.92 ng/ml, respectively.

The retina choroid was homogenized and analyzed as described in Example3, with the samples taken as described for the vitreous above. Theretina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 5, 30,60, 90, and 120 days after subconjunctival injection was about 0.47,0.04, 0.01, 0.05, and 0.0 ng/mg, respectively.

Example 5 Subconjunctival Injection of a Rapamycin-Containing Solution

60 μl of the solution described in Example 23 were injected between thesclera and the conjunctiva of the eye of New Zealand white rabbits. FIG.2 depicts the level of rapamycin in the vitreous on a logarithmic scaleat 5, 30, 60, 90, and 120 days after injection. FIG. 3 depicts the levelof rapamycin in the retina choroid on a logarithmic scale at the sametime points.

The vitreous was homogenized and analyzed as described in Example 3.Between two and five rabbit eyes were analyzed at each time point. Thevitreous sample did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the vitreous.The average level of rapamycin in the vitreous at 5, 30, 60, 90, and 120days after subconjunctival injection was about 8.65, 0.29, 0.18, 2.00,1.41 ng/ml, respectively.

The retina choroid was homogenized and analyzed as described in Example3, with the samples taken as described for the vitreous above. Theretina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 5, 30,60, 90, and 120 days after subconjunctival injection was about 0.63,0.02, 0.02, 0.06, and 0.01 ng/mg, respectively.

Example 6 Intravitreal Injection of a Rapamycin-Containing Solution

20 μl of the solution described in Example 2 were injected into thevitreous of the eye of New Zealand white rabbits. The injected solutionformed a non-dispersed mass relative to the surrounding medium. FIG. 4depicts the level of rapamycin in the vitreous on a logarithmic scale 5,30, 60, 90, and 120 days after injection. FIG. 5 depicts the level ofrapamycin in the retina choroid on a logarithmic scale at the same timepoints. For comparison, FIG. 4 and FIG. 5 also depict results of otherstudies described below in Example 28 and Example 30.

The vitreous was homogenized and analyzed as described in Example 3.Between two and five rabbit eyes were analyzed at each time point. Thevitreous sample may have included the site of administration. Theaverage level of rapamycin in the vitreous at 5, 30, 60, 90, and 120days after intravitreal injection was about 162,100; 18,780; 57,830;94,040; and 13,150 ng/ml, respectively.

The retina choroid was homogenized and analyzed as described in Example3, with the samples taken as described for the vitreous above. Theretina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 5, 30,60, 90, and 120 days after intravitreal injection was about 2.84, 2.26,0.17, 0.22, and 0.05 ng/mg, respectively.

Example 7 Intravitreal Injection of a Rapamycin-Containing Solution

40 μl of the solution described in Example 2 were injected into thevitreous of the eye of New Zealand white rabbits. The injected solutionformed a non-dispersed mass relative to the surrounding medium. FIG. 4depicts the level of rapamycin in the vitreous on a logarithmic scale 5,30, 60, 90, and 120 days after injection. FIG. 5 depicts the level ofrapamycin in the retina choroid on a logarithmic scale at the same timepoints.

The vitreous was homogenized and analyzed as described in Example 3.Between two and five rabbit eyes were analyzed at each time point. Thevitreous sample may have included the site of administration. Theaverage level of rapamycin in the vitreous at 5, 30, 60, 90, and 120days after intravitreal injection was about 415,600; 4,830; 74,510;301,300; and 7,854 ng/ml respectively.

The retina choroid was homogenized and analyzed as described in Example3, with the samples taken as described for the vitreous above. Theretina choroid did not include the site of administration, so thismeasurement indicated the level of rapamycin delivered to the retinachoroid. The average level of rapamycin in the retina choroid at 5, 30,60, 90, and 120 days after intravitreal injection was about 5.36, 0.23,1.27, 1.08, and 0.08 ng/mg, respectively.

All references cited herein, including patents, patent applications, andpublications, are hereby incorporated by reference in their entireties,whether previously specifically incorporated or not.

What is claimed is:
 1. A method for delaying progression of dryage-related macular degeneration to wet age-related macular degenerationin a human subject with dry age-related macular degeneration, the methodcomprising administering to the human subject by intraocular orperiocular injection a volume of a liquid formulation containing anamount of rapamycin effective to delay progression of dry-age maculardegeneration to wet age-related macular degeneration in the humansubject, wherein the liquid formulation is a liquid solution thatcomprises about 2% (w/w) rapamycin, about 94% (w/w) PEG 400, and about4% (w/w) ethanol.
 2. The method of claim 1, wherein the volume of liquidformulation contains between 20 μg and 2.5 mg of rapamycin.
 3. Themethod of claim 1, wherein the volume of liquid formulation containsbetween 20 μg and 4 mg of rapamycin.
 4. The method of claim 1, whereinthe volume of the liquid formulation is administered to the humansubject by injection into the vitreous.
 5. The method of claim 1,wherein the volume of the liquid formulation is administered to thehuman subject by injection between the sclera and conjunctiva.
 6. Themethod of claim 1, wherein the volume of the liquid formulation wheninjected into the vitreous delivers an amount of rapamycin sufficient toachieve one or both of: an average concentration of rapamycin in theretina choroid of at least 0.01 ng/mg for a period of time of at least30 days following administration of the liquid formulation, and anaverage concentration of rapamycin in the vitreous of at least 1000ng/ml for a period of time of at least 30 days following administrationof the liquid formulation.
 7. The method of claim 1, wherein the volumeof the liquid formulation when injected between the sclera andconjunctiva delivers an amount of rapamycin sufficient to achieve one orboth of: an average concentration of rapamycin in the vitreous of atleast 0.01 ng/ml for a period of time of at least 30 days followingadministration of the liquid formulation; and an average concentrationof rapamycin in the retina choroid of at least 0.001 ng/mg for a periodof time of at least 30 days following administration of the liquidformulation.